Isolated P27 protein, nucleic acid molecules encoding same, methods of identifying agents acting on same, and uses of said agents

ABSTRACT

An isolated protein designated p27 is disclosed. The p27 protein has an apparent molecular weight of about 27 kD, and is capable of binding to and inhibiting the activation of a cyclin E-Cdk2 complex. A nucleic acid sequence encoding p27 protein is disclosed, as well as a method for producing p27 in cultured cells. in vitro assays for discovering agents which effect the activity of p27 are also provided. Methods of diagnosing and treating hypoproliferative and hyperproliferative disorders are provided.

RELATED U.S. APPLICATION(S)

This application is a national stage filing of PCT Application No.PCT/US95/07361, filed Jun. 7, 1995, now U.S. Pat. No. 5,688,665, whichis a continuation-in-part of U.S. application Ser. No. 08/275,983, filedJul. 15, 1994.

This invention was made with support under Grant No. CA48718 from theNational Institutes of Health. Accordingly, the U.S. government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced. Fullcitations for these references may be found at the end of thespecification immediately preceding the claims. The disclosures of thesepublications are hereby incorporated by reference into this applicationto describe more fully the art to which this invention pertains.

Progression through the cell cycle is marked by a series of irreversibletransitions that separate discrete tasks necessary for faithful cellduplication. These transitions are negatively regulated by signals thatconstrain the cell cycle until specific conditions are fulfilled. Entryinto mitosis, for example, is inhibited by incompletely replicated DNAor DNA damage (Weinert and Hartwell, 1988). Another feedback pathwaydelays the transition from M to G1 if the mitotic spindle is defective(Hoyt et al., 1991; Li & Murray, 1991). These restrictions on cell cycleprogression are essential for preserving the fidelity of the geneticinformation during cell division (Hartwell & Weinert, 1989). Thetransition from G1 to S phase, on the other hand, coordinates cellproliferation with environmental cues, after which the checks on cellcycle progression tend to be cell autonomous (Hartwell et al., 1974;Pardee 1974, 1989). Among the extracellular influences that restrictcell cycle progression during G 1 are proteins that inhibit cellproliferation, growth factor or amino acid depletion, and cell-cellcontact. Disruption of these signaling pathways uncouples cellularresponses from environmental controls and may lead to unrestrained cellproliferation.

Transitions between phases of the cell cycle are catalyzed by a familyof cyclin-dependent kinases (Cdks) (Nurse, 1990; Hartwell, 1991). Insome organisms the physiological signals controlling the G2 to Mtransition target a series of steps that activate the mitotic Cdk, Cdc2.Cdc2 activation is positively regulated by phosphorylation onthreonine-161 (Booher & Beach, 1986; Krek & Nigg, 1991; Gould et al.,1991; Solomon et al., 1990; 1992) and negatively by phosphorylation ontyrosine-15 (Gould & Nurse, 1989). Incomplete DNA replication delaysdephosphorylation of tyr-15 (Dasso & Newport, 1990; Smythe & Newport,1992), and mutations in Cdc2 that convert tyr-15 to anonphosphorylatable residue are lethal and cause a premature mitosis(Gould & Nurse, 1989). Similarly, either over expression of the tyr-15phosphatase, Cdc25 (Enoch & Nurse, 1990; Kumagai & Dunphy, 1991), orloss of the tyr-15 kinases (Ludgren et al., 1991) bypass the requirementthat DNA replication be completed before mitosis begins. Additionallevels of control are probably required to fully explain the block tomitosis caused by ongoing DNA replication (Sorger & Murray, 1992; Healdet al., 1993; Stueland et al., 1993). There is also evidence that cellcycle arrest induced by DNA damage may be related to inactivation ofCdc2 (Rowley et al., 1992; Walworth et al., 1993), but the role oftyrosine phosphorylation in this context has been questioned (Barbet &Carr, 1993).

There is some evidence, particularly in yeast, that signals inhibitingthe G1 to S phase transition block Cdk activation. The mating pheromonealpha factor arrests the S. cerevisiae cell cycle in G1 (Reid &Hartwell, 1977), and this correlates with a decrease in CDC28 kinaseactivity and a decline in the abundance of active complexes containingG1 cyclins and CDC28 (Wittenberg et al., 1990). The FARl protein bindsto G1 cyclin-CDC28 complexes in cells treated with alpha factor, andthis is probably necessary for cell cycle arrest (Chang & Herskowitz,1990; Peter et al., 1993). Other inhibitors of CDC28 kinase activityhave been identified, but their relationship to physiological signalsthat control cell cycle progression is not known (Mendenhall, 1993;Dunphy & Newport, 1989).

Mammalian cells, like yeast, require cyclin-dependent kinases forprogression through G1 and entry into S phase (D'Urso et al., 1990; Blow& Nurse, 1990; Furukawa et al., 1990; Fang & Newport, 1991; Pagano etal., 1993; Tsai et al., 1993). Both D and E-type cyclins are ratelimiting for the G1 to S transition and both reduce, but do noteliminate, the cell's requirement for mitogenic growth factors (Ohtsubo& Roberts, 1993; Quelle et al., 1993). There is little information,however, concerning the manner by which these cyclins and Cdks arenegatively regulated by extracellular signals that inhibit cellproliferation.

It has been studied how two signals that block the cell cycle in G1,cell-cell contact and TGF-β, affect the activity of a G1cyclin-dependent kinase, Cdk2 (Paris et al., 1990; Elledge & Spotswood,1991; Koff et al., 1991; Tsai et al., 1991; Elledge et al., 1992;Rosenblatt et al., 1992). The cell cycle of Mv1Lu mink epithelial cellscan be arrested in G1 by growth to high density. These contact inhibitedcells express both cyclin E and Cdk2, but cyclin E-associated kinaseactivity is not present (Koff et al., 1993). Entry into S phase can alsobe prevented if Mv1Lu cells are released from contact inhibition in thepresence of TGF-β, and this correlates with a block to phosphoryla-tionof the Retinoblastoma (Rb) protein (Laiho et al. 1990). Both Cdk2 andCdk4 have been implicated as Rb kinases (Matsushime et al., 1992; Hindset al., 1993; Kato et al., 1993; Ewen et al., 1993a; Dowdy et al.,1993), suggesting that TGF-β induced cell cycle arrest may involveinhibition of Cdks during G1 (Howe et al., 1991). Consistent with this,cells arrested in late G1 by TGF-β, like contact inhibited cells,express both cyclin E and Cdk2 but do not contain catalytically activecyclin E-Cdk2 complexes (Koff et al., 1993). Cdk4 synthesis is alsorepressed by TGF-β (Ewen et al., 1993b). The inactivity of Cdk2 togetherwith the absence of Cdk4 may explain the block to Rb phosphorylation inthese cells.

It is shown herein that contact inhibited and TGF-β treated cells, butnot proliferating cells, contain a titratable excess of a 27 kD proteinthat binds to the cyclin E-Cdk2 complex and prevents its activation. Theinhibitory activity of p27 can be competed by the cyclin D2-Cdk4complex, suggesting that p27 and cyclin D2-Cdk4 may function within apathway that transmits growth inhibitory signals to Cdk2.

The subject invention provides an isolated 27 kD protein capable ofbinding to and inhibiting the activation of a cyclin E-Cdk2 complex. Thesubject invention further provides related recombinant nucleic acidmolecules, host vector systems and methods for making same. Finally, thesubject invention provides methods of identifying agents and usingagents which act on or mimic p27 function, so as to exploit theregulatory role of p27 in cell proliferation.

SUMMARY OF THE INVENTION

The subject invention provides an isolated protein having an apparentmolecular weight of about 27 kD as measured by SDS polyacrylamide gelelectrophoresis, and capable of binding to and inhibiting the activationof a cyclin E-Cdk2 complex.

The subject invention further provides a recombinant nucleic acidmolecule which encodes the protein of the subject invention.

The subject invention further provides a vector comprising therecombinant nucleic acid molecule of the subject invention.

The subject invention further provides a host vector system for theproduction of a protein having an apparent molecular weight of about 27kD as measured by SDS polyacrylamide gel electrophoresis, and capable ofbinding to and inhibiting the activation of a cyclin E-Cdk2 complex,which comprises the vector of the subject invention in a suitable host.

The subject invention further provides a method for producing a proteinhaving an apparent molecular weight of about 27 kD as measured by SDSpolyacrylamide gel electrophoresis, and capable of binding to andinhibiting the activation of a cyclin E-Cdk2 complex, which comprisesgrowing the host vector system of the subject invention under conditionspermitting the production of the protein and recovering the proteinproduced thereby.

The subject invention further provides a method of determining whetheran agent is capable of specifically inhibiting the ability of p27protein to inhibit the activation of cyclin E-Cdk2 complex whichcomprises: (a) contacting suitable amounts of p27 protein, cyclin E,Cdk2 and the agent under suitable conditions; (b) subjecting the p27,cyclin E, Cdk2, and agent so contacted to conditions which would permitthe formation of active cyclin E-Cdk2 complex in the absence of p27protein; (c) quantitatively determining the amount of active cyclinE-Cdk2 complex so formed; and (d) comparing the amount of active cyclinE-Cdk2 complex so formed with the amount of active cyclin E-Cdk2 complexformed in the absence of the agent, a greater amount of active cyclinE-Cdk2 complex formed in the presence of the agent than in the absenceof the agent indicating that the agent is capable of specificallyinhibiting the ability of p27 protein to inhibit the activation ofcyclin E-Cdk2 complex.

The subject invention further provides a method of determining whetheran agent is capable of specifically enhancing the ability of p27 proteinto inhibit the activation of cyclin E-Cdk2 complex which comprises: (a)contacting suitable amounts of p27 protein, cyclin E, Cdk2 and the agentunder suitable conditions; (b) subjecting the p27, cyclin E, Cdk2, andagent so contacted to conditions which would permit the formation ofactive cyclin E-Cdk2 complex in the absence of p27 protein; (c)quantitatively determining the amount of active cyclin E-Cdk2 complex soformed; and (d) comparing the amount of active cyclin E-Cdk2 complex soformed with the amount of active cyclin E-Cdk2 complex formed in theabsence of the agent, a lesser amount of active cyclin E-Cdk2 complexformed in the presence of the agent than in the absence of the agentindicating that the agent is capable of specifically enhancing theability of p27 protein to inhibit the activation of cyclin E-Cdk2complex.

The subject invention further provides a method of treating a subjecthaving a hyperproliferative disorder which comprises administering tothe subject a therapeutically effective amount of an agent capable ofspecif ically enhancing the ability of p27 protein to inhibit theactivation of cyclin E-Cdk2 complex in the hyperproliferative cells ofthe subject, so as to thereby treat the subject.

The subject invention further provides a method of treating a subjecthaving a hypoproliferative disorder which comprises administering to thesubject a therapeutically effective amount of an agent capable ofspecifically inhibiting the ability of p27 protein to inhibit theactivation of cyclin E-Cdk2 complex in the hypoproliferative cells ofthe subject, so as to thereby treat the subject.

The subject invention further provides a method of diagnosing ahyperproliferative disorder in a subject which disorder is associatedwith the presence of a p27 protein mutation in the cells of the subject,which comprises determining the presence of a p27 protein mutation inthe cells of the subject, said mutation being associated with ahyperproliferative disorder, so as to thereby diagnose ahyperproliferative disorder in the subject.

The subject invention further provides a pharmaceutical compositionwhich comprises an effective amount of a recombinant virus capable ofinfecting a suitable host cell, said recombinant virus comprising thenucleic acid molecule of the subject invention, and a pharmaceuticallyacceptable carrier.

Finally, this invention provides a method for treating a subjectsuffering from a hyperproliferative disorder associated with thepresence of a p27 protein mutation in the cells of the subject, whichcomprises administering to the subject an amount of the pharmaceuticalcomposition of the subject invention effective to treat the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A

Activation of Cdk2 by cyclin E in extracts from proliferating and growtharrested cells. Cyclin E was added to extracts from contact inhibitedcells (0), and cells released from contact inhibition for 15 hours inthe presence (0β15) or absence (15) of TGF-B. The 15 hour cells arereferred to in the text as “proliferating cells” to indicate that theyare progressing through the cell cycle and have entered S phase. 0.05 μlof cyclin E corresponds to physiological levels of cyclin E in theseextracts. The inset shows the titration of up to 3 times physiologicallevels of cyclin E. Cyclin E immunoprecipitates were assayed for histoneH1 kinase activity and the results quantitated using a phosphorimager.Background levels of phosphorylation observed in the absence ofexogenous cyclin E were subtracted from each sample.

FIG. 1B

Activation of Cdk2 by cyclin E in extracts from proliferating and growtharrested cells. Extracts were prepared from contact inhibited cells (0),and cells released from contact inhibition for 15 hours in the presence(0β15) or absence (15) of TGF-β. Physiological amounts of cyclin E wereadded to various amounts of these extracts and to mixtures of extracts.Extracts from proliferating and arrested cells were mixed in theindicated proportions, and the total amount of protein in each mixturewas 75 μg. The amount of each extract used (μg) is indicated. Afterincubation, cyclin E was immunoprecipitated and assayed for H1 kinaseactivity. Results were quantitated using a phosphorimager. Backgroundlevels of phosphorylation observed in the absence of exogenous cyclin Ewere subtracted from each sample.

FIG. 2A

A Cdk2 inhibitor binds to cyclin E-Cdk2 complexes. The indicatedextracts were incubated with a Cdk2-sepharose beads (K), cyclin E-Cdk2sepharose beads (EK), or blank sepharose beads (0). The Cdk2 beadscontained 2-fold more Cdk2 than present in the cell extract. The cyclinE-Cdk2 beads contained approximately 60-fold more cyclin E than waspresent in the cell extract. After incubation a portion of eachsupernatant was analyzed by Western blotting to confirm that neithercyclin E nor CDK2 had leached from the matrices. The remainder of thesupernatant was assayed for Cdk2 activation by addition of 2×physiological amounts of cyclin E. Cyclin E immunoprecipitates wereassayed for H1 kinase activity and the results quantitated using aphsophorimager. Partial depletion of inhibitor by the Cdk2 beads may beattributable to the formation of cyclin-Cdk2 complexes during theincubation with the cell extract.

FIG. 2B

A Cdk2 inhibitor binds to cyclin E-Cdk2 complexes. Cdk2 wasimmunoprecipitated from extracts of contact inhibited cells (0), andcells released from contact inhibition for 15 hours in the presence(0β15) or absence (15) of TGF-β. Half of each immunoprecipitate wasincubated with cyclin E plus CAK, and the other half underwent mockincubation. Each immuno-precipitate was then assayed for histone H1kinase activity and the results quantitated using a phosphorimager. Inthe absence of added CAK, cyclin E had only a very small activatingeffect on immunoprecipitated Cdk2 (data not shown).

FIG. 2C

A Cdk2 inhibitor binds to cyclin E-Cdk2 complexes. Effect of kinaseinactive Cdk2 on cyclin E activity in extracts from growth arrestedcells. Each extract was incubated with 5 fold excess of cyclin E (justat the cyclin E threshold for this lysate), 0.5 fold excess of kinaseinactive Cdk2 (Cdk2K), or both. These proportions were chosen based uponempirical determinations of the maximum amount of Cdk2K that could beadded without sequestering the majority of the added cyclin E. Cyclin Eimmunoprecipitates were assayed for H1 kinase activity and the resultsquantitated using a phosphorimager.

FIG. 3A

Activation of Cdc2 by cyclin B. Cyclin B and Cyclin E were added toextracts from cells released from contact inhibition for 15 hours in thepresence (0β15) or absence (15) of TGF-β. After addition of cyclins theextracts were divided and immunoprecipitated with either antiseradirected to the C-terminus of Cdc2 or the C-terminus of Cdk2. Theimmunoprecipitates were assayed for H1 kinase activity and the productsresolved on a 12% polyacrylamide gel. The reactions labeled “endogenous”contain no added cyclin.

FIG. 3B

Activation of Cdc2 by cyclin B. Cyclin B was added to extracts fromcells released from contact inhibition for 15 hours in the presence(0β15) or absence (15) of TGF-β. Half of each reaction was supplementedwith purified CAK. Cdc2 was immunoprecipitated with antibody directedtowards the C-terminus of Cdc2 and assayed for H1 kinase activity. Theresults were quantitated using a phosphorimager.

FIG. 4A

Effect of cyclin D-Cdk4 complexes on cyclin E activity. Extracts wereprepared from contact inhibited cells (0), and cells released fromcontact inhibition for 15 hours in the presence (0β15) or absence (15)of TGF-β. 0.05 microliters of Sf9 cell lysates containing cyclin D2,Cdk4, cyclin D2-Cdk4 complexes, or complexes containing cyclin D2 boundto catalytically inactive Cdk4 (Cdk4K) were added to these extractstogether with physiological amounts of cyclin E. These amounts of cyclinD2 and Cdk4 closely correspond to physiological amounts of theseproteins. Cyclin E was immunoprecipitated and assayed for associatedhistone H1 kinase activity.

FIG. 4B

Effect of cyclin D-Cdk4 complexes on cyclin E activity. Extracts wereprepared from cells released from contact inhibition for 15 hours in thepresence (0β15) or absence (15) of TGF-β. 0.05 microliters of Sf9 celllysates containing the indicated cyclin D-Cdk4 complexes were added tothese extracts in the presence of absence of cyclin E. Cyclin E wasimmunoprecipitated and assayed for associated histone H1 kinaseactivity.

FIG. 5A

A 27 kD cyclin E-Cdk2 binding protein. Contact inhibited Mv1Lu cellswere released from quiescence by replating at lower density and extractsprepared from 35S-methionine labeled cells at 0 and 15 h. Some cultureswere incubated in the presence of 100 pM TGF-B for 15 h (0β15). Thesemetabolically labeled extracts were treated as described and boundproteins eluted in sample buffer and analyzed by SDS-PAGE followed byfluorography. Migration of molecular weight markers are shown.35S-methionine labeled cell extracts were incubated with Cdk2 or cyclinE-Cdk2 complexes and bound proteins eluted in sample buffer. The arrowindicates the migration of a 27 kD protein (p27) specifically associatedwith cyclin E-Cdk2 complexes in extracts from contact inhibited andTGF-β treated cells.

FIG. 5B

A 27 kD cyclin E-Cdk2 binding protein. Extracts from metabolicallylabeled contact inhibited Mv1Lu cells were incubated with varyingamounts of Cdk2 or cyclin E-Cdk2 relative to standard conditions andbound proteins analyzed as described below. The presence of p27 isindicated (arrow).

FIG. 5C

A 27 kD cyclin E-Cdk2 binding protein. Cyclin D2-Cdk4 complexes preventbinding of p27 to cyclin E-Cdk2. Extracts from contact inhibited cellswere preincubated with 4 μl of baculovirus produced cyclin D2-Cdk4complex for 30 minutes at 4° C. prior to addition of the cyclin E-Cdk2complex.

FIG. 5D

A 27 kD cyclin E-Cdk2 binding protein. Recovery of p27 in Cdk4immunoprecipitates. Supernatants from 5C were immunoprecipitated with ananti-Cdk4 antiserum and immunoprecipitates were analyzed on 12%SDS-PAGE. The open arrow at 34 kD shows the endogenous mink Cdk4protein, while the closed arrow indicates p27, associated with thecyclin D2-Cdk4 complexes.

FIG. 6A

Heat stability of D27 and the Cdk2 inhibitor. p27 binding is heat stableand p27 call be recovered from proliferating cell extracts by heattreatment. Mv1Lu cells were released from contact inhibition for 15hours with (0β15) or without (15) TGF-βl. Cells were metabolicallylabeled using 35S-methionine. Prior to incubation with Cdk2 or cyclinE-Cdk2 complexes cell extracts received either no pretreatment or wereheated to 100° C. for 3 minutes. Note the appearance of p27 (arrow) inheat treated 15h cell extract.

FIG. 6B

Heat stability of D27 and the Cdk2 inhibitor. Cdk2 inhibitory activitycan be recovered from proliferating cell extracts by heat treatment.Cyclin E associated kinase activity was measured in extracts fromasynchronous proliferating cells by immunoprecipitation with antibodiesagainst human cyclin E. Histone H1 was the substrate and results werequantitated using a phosphorimager. Lane 1—no additions; Lane 2—theextract was supplemented with 3 times physiological amounts of cyclin E;Lane 3—as in lane 2 except that heat treated extract from proliferatingcells (see methods) was also added to the cell extract.

FIG. 6C

Heat stability of p27 and the Cdk2 inhibitor. Cdk2 inhibitory activitywas heat stable. Extracts were prepared from contact inhibited cells(0), cells released from contact inhibition for 48 hours in the presenceof TGF-β (0β48) or asynchronous proliferating cells (Exp). Cyclin Eassociated kinase activity measured with or without addition ofexogenous cyclin E. In the indicated lanes proliferating cell extractswere mixed with an equal amount of extract from nonproliferating cells,that had either been untreated or heated to 100° C. for 5 minutes.

FIG. 7A

Inhibition of cyclin E-associated kinase activity by purified p27.Extracts from metabolically labeled contact inhibited Mv1Lu cells weresubjected to chromatography on Cdk2 or cyclin E-Cdk2 affinity columns.Bound proteins, eluted in low pH buffer, and were analyzed by SDS-PAGE.p27 present in cyclin E-Cdk2 eluates is shown (arrow).

FIG. 7B

Eluates from Cdk2 or cyclin E-Cdk2 columns were recipitated with acetoneand renatured (see methods). A ortion of each eluate was added to anextract from proliferating cells. Cyclin E was added and cyclin Eassociated histone H1 kinase activity measured. The cyclin E associatedH1 kinase activity was quantitated and plotted as % inhibition relativeto extracts receiving no additions.

FIG. 7C

Renatured eluates were incubated with Cdk2 or cyclin E-Cdk2 complexes.p27 (arrow) bound to cyclin E-Cdk2 after renaturation.

FIG. 7D

Eluates from cyclin E-cdk2 columns were fractionated on 12% acrylamidegels. The gels were sliced as shown and proteins were eluted andrenatured. A portion of the protein recovered from each gel slice wasadded together with cyclin E to extracts prepared from proliferatingMv1Lu cells. Cyclin E immunoprecipitates were assayed for histone H1kinase activity.

FIGS. 8A, 8B, 8C and 8D

Purification, cyclin E-Cdk2 interaction, and in vitro translation ofKip1. A, Heat-treated extracts from quiescent Mv1Lu cells were subjectedto cyclin E-Cdk2 affinity chromatography. The eluate was resolved bySDS-PAGE and silver stained. p27^(Kip1) is indicated by an arrow. Thebroad band is Cdk2-HA, and the 69 kd band is a contaminant present alsoin blank lanes. B, Extracts from metabolically labeled quiescent Mv1Lucells were precipitated with preimmune rabbit serum (control) oranti-Cdk2 antibody. C. metabolically-labeled p27 obtained bycoprecipitation with anti-Cdk2 antibodies as in panel B (in vivo p27) orby cyclin E-Cdk2 affinity chromatography as in panel A (in vitro p27)was digested with V8 protease and displayed by SDS-PAGE andfluorography. D, In vitro translations containing empty vector (vector)or vector encoding histidine-tagged mouse Kip1 (Kip1) were bound toNi⁺⁺-NTA-agarose, boiled in ample buffer and resolved by SDS-PAGE.

FIGS. 9A and 9B

Mammalian Kip1 sequences, and comparison with Cip1/WAF1. A, Amino acidsequences deduced from Kip1 cDNAs from mink (mk), mouse (m) and human(h). Identical amino acids are indicated by dots. The available minksequence is incomplete at the C-terminus. Peptide sequences obtainedfrom purified Kip1 are underlined. Thick underlining indicates the twosequences that served to design degenerate oligonucleotides for PCR. Thefirst occurrence of thick underlining is directed to mink kip1 and mousekip1 sequence NLFGPVNHEELTR (SEQ ID NO: 26) and human kip1 sequenceNLFGPVDHEELTR (SEQ ID NO: 27). The sequence LFGPVN (SEQ ID NO: 22)within NLFGPVNHEELTR and the sequence LFGPVD (SEQ ID NO: 25) withinNLFGPVDHEELTR respectively correspond to the longest uninterruptedstretch of identity to Cip1/WAF1 (SEQ ID NO: 24). B, Sequence alignmentbetween human Kip1 and Cip1/WAF1. The putative bipartite nuclearlocalization signal in both proteins is underlined. A Cdc2 kinaseconsensus site present in Kip1 is indicated by a thick bar.

FIGS. 10A, 10B, 10C, 10D and 10E

Cdk inhibition by Kip1 in vitro, and identification of an Cdk inhibitorydomain of Kip1. Cell lysates containing baculoviral cyclin E and Cdk2(A, C) or the indicated cyclin/Cdk combinations, were assayed forhistone H1 kinase activity (A, B) and Rb kinase activity (C, D) in thepresence of the indicated concentrations of Kip1. Representative gelscontaining the phosphorylated substrates are shown (A, C). Relativephosphorylation levels were quantitated, and are plotted as thepercentage of phosphorylation observed in reactions without Kip1. E,Schematic of the Kip1 protein indicating the regions of highest homologyto Cip1/WAF1 (shaded boxes; see also FIG. 9B). Bars and numbers indicatethe size and location of the various fragments produced with aC-terminal hexahistidine tag and used in Cdk inhibition assays. Theactivity of these fragments is presented as a percentage relative to theactivity of full length Kip1.

FIGS. 11A and 11B

Kip1 inhibits activation of Cdk2 in vitro. Extracts from exponentiallygrowing A549 cells where incubated with baculovirally expressedhistidine-tagged cyclin E alone or together with Kip1. Cyclin Ecomplexes were then retrieved with Ni⁺⁺-NTA-agarose, and assayed forhistone H1 kinase activity (A), and by western immunoblotting usinganti-Cdk2 antibody (B). Kinase activity was quantitated byPhosphorimager and expressed as arbitrary units. In B, Cdk2* indicatesthe faster migrating form of Cdk2 that corresponds to Cdk2phosphorylated at Thr¹⁶⁰ (Gu et al., 1992).

FIGS. 12A and 12B

Expression pattern of Kip1 in various tissues and cell proliferationstates. Kip1 Northern blots using equal amounts of poly(A)⁺ RNA from theindicated human tissues (A) or from Mv1Lu cells in differentproliferation states (B). The latter blot was rehybridized with aglyceraldehyde-phosphate dehydrogenase probe.

FIGS. 13A and 13B

Mink Kip1 cDNA and the encoded mink kip1 (SEQ ID NOs: 5 and 6)

FIGS. 14A and 14B

Mouse Kip1 cDNA and the encoded mouse kip1 (SEQ ID NOs: 3 and 4)

FIGS. 15A and 15B

Human Kip1 cDNA and the encoded human kip1 (SEQ ID NOs: 1 and 2)

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides an isolated protein having an apparentmolecular weight of about 27 kD as measured by SDS polyacrylamide gelelectrophoresis, and capable of binding to and inhibiting the activationof a cyclin E-Cdk2 complex.

In the subject invention, the SDS polyacrylamide gel electrophoresisused to obtain the 27 kD molecular weight is performed under reducingconditions.

In one embodiment, the isolated protein of the subject invention is amammalian protein. The mammalian protein may be a murine protein. Themammalian protein may also be a human protein. The mammalian protein mayfurther be a mink protein. In one embodiment, the mink protein is themink protein derived from Mv1Lu cells and having the partial internalamino acid sequences shown in Table I.

TABLE I Partial Internal Amino Acid Sequences of Mv1Lu Cell-Derived d27Protein 1. Asn-Leu-Tyr-Pro-Leu-Asn-Tyr-Thr-Phe SEQ ID NO. 7 2.Thr-Asp-Thr-Ala-Asp-Asn-Gln-Ala-Gly-Leu-Ala-Glu-Gln SEQ ID NO. 8 3.Gln-Ala-Val-Pro-Leu-Met-Gly-Pro-Gln-Glu SEQ ID NO. 9 4.Leu-Pro-Glu-Phe-Tyr-Tyr-Arg-Pro-Pro-Arg-Pro-Pro SEQ ID NO. 10 5.Tyr-Glu-Trp-Gln-Glu-Val SEQ ID NO. 11

In the subject invention, the protein having an apparent molecularweight of about 27 kD as measured by SDS polyacrylamide gelelectrophoresis, and capable of binding to and inhibiting the activationof a cyclin E-Cdk2 complex, is referred to synonymously as “p27”, “p27protein”, “inhibitor”, “p27^(Kip1)” and “Kip1”.

As used herein, “isolated” means free of any other proteins. Forexample, the isolated protein may include nitrocellulose membranefragments and a buffer.

As used herein, “capable of binding to a cyclin E-Cdk2 complex” meanscapable of binding to a cyclin E-Cdk2 complex but incapable of bindingto Cdk2 alone.

Inhibition of the activation of a cyclin E-Cdk2 complex may be measured,for example, using assays for (a) the site-specific phosphorylation ofthe Cdk2 moiety of the cyclin E-Cdk2 complex and (b) histone kinaseactivity. Such assays are discussed in more detail infra. These assaysmay be conducted in a kinetic mode (i.e., by measuring the rate ofphosphorylation) or as qualitative or quantitative static assays (i.e.,measurements made at selected points in time). Those skilled in the artwill know that a variety of enzymes and conditions may be used in suchassays. In the subject invention, in a kinetic mode assay usingequimolar amounts of p27 and cyclin E-Cdk2 complex, p27 inhibits therate of site-specific phosphorylation of the Cdk2 moiety of the complex(as expressed in moles of Cdk2 moiety phosphorylated per minute) if therate is inhibited by at least 50%.

The isolated 27 kD protein of the subject invention may be obtained, byway of example, by the heat treatment method and by the cyclin E-Cdk2complex affinity method described infra.

The subject invention further provides a protein comprising a portionhaving amino acid sequence homology with the portion of p27 protein fromamino acid residue +28 to, and including, amino acid residue +88 (asshown in FIG. 9B). In one embodiment, the protein has sequence identitywith at least one boxed amino acid residue in the portion of p27 proteinfrom amino acid residue +28 to, and including, amino acid residue +88(as shown in FIG. 9B). The protein may be naturally occurring orrecombinant. In one embodiment, the degree of homology is 30%. Inanother embodiment, the degree of homology is 40%. In anotherembodiment, the degree of homology is 44%. In another embodiment, thedegree of homology is 50%. In another embodiment, the degree of homologyis 90%.

The subject invention further provides recombinant nucleic acidmolecules which encodes the proteins of the subject invention.

As used herein, a recombinant nucleic acid molecule is a nucleic acidmolecule which does not occur in nature and which is obtained throughthe use of recombinant technology.

In one embodiment, the nucleic acid molecule is a DNA molecule. The DNAmolecule may be a cDNA molecule or a cloned genomic DNA molecule. In afurther embodiment, the cDNA is a mink kip1 cDNA. In a still furtherembodiment, the nucleotide sequence of mink kip1 cDNA is substantiallythe same as described in FIGS. 13A and 13B.

In a separate embodiment, the cDNA is a mouse kip1 cDNA. In a furtherembodiment, the nucleotide sequence of mouse kip1 cDNA is substantiallythe same as described in FIGS. 14A and 14B.

In a still separate embodiment, the cDNA is a human kip1 cDNA. In afurther embodiment, the nucleotide sequence of human kip1 cDNA issubstantially the same as described in FIGS. 15A and 15B.

In another embodiment, the nucleic acid molecule is an RNA molecule. TheRNA molecule may be an mRNA molecule.

The subject invention further provides a vector comprising therecombinant nucleic acid molecule of the subject invention. In oneembodiment, the vector is a plasmid. In another embodiment, the vectoris a virus.

In a specific embodiment, a human kip1 cDNA with 2780 nucleotidescontaining 5′untranslated region, coding sequence and stop codon iscloned between KpnI and BamHI sites within the polylinker of the pCMV5vector. This plasmid is designated pCMV5 p27kip1.

The, pCMV5 p27kip1 was deposited on Jun. 7, 1995 with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852,U.S.A. under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganism for the Purposes of PatentProcedure. Plasmid pCMV5 p27kip1 was accorded ATCC accession number97203.

For the purpose of illustration only, applicants have isolated andcharacterized kip1 cDNA clones from human and mouse cDNA library using amink kip1 cDNA. See infra. Similarly, other mammalian kip1 may beisolated using the known kip1 cDNAs disclosed in this invention.Briefly, the homologous genes may be cloned by using probe from themink, mouse, or human kip1 cDNA by low stringency screening of thecorrespondent cDNA libraries.

In accordance with the invention, numerous vector systems for expressionof the protein of the subject invention may be employed. For example,one class of vectors utilizes DNA elements which are derived from animalviruses such as bovine papilloma virus, polyoma virus, adenovirus,vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV), SemlikiForest virus or SV40 virus. Additionally, cells which have stablyintegrated the DNA into their chromosomes may be selected by introducingone or more markers which allow for the selection of transfected hostcells. The marker may provide, for example, prototropy to an auxotrophichost, biocide resistance, (e.g., antibiotics) or resistance to heavymetals such as copper or the like. The selectable marker gene can beeither directly linked to the DNA sequences to be expressed, orintroduced into the same cell by cotransformation. Additional elementsmay also be needed for optimal synthesis of mRNA. These elements mayinclude splice signals, as well as transcriptional promoters, enhancers,and termination signals.

The subject invention further provides a host vector system for theproduction of a protein having an apparent molecular weight of about 27kD as measured by SDS polyacrylamide gel electrophoresis, and capable ofbinding to and inhibiting the activation of a cyclin E-Cdk2 complex,which comprises the vector of the subject invention in a suitable host.

In one embodiment, the suitable host is a bacterial cell. In anotherembodiment, the suitable host is an eucaryotic cell. The eucaryotic cellmay be an insect cell. Insect cells include, by way of example, sf9cells.

The subject invention further provides a method for producing a proteinhaving an apparent molecular weight of about 27 kD as measured by SDSpolyacrylamide gel electrophoresis, and capable of binding to andinhibiting the activation of a cyclin E-Cdk2 complex, which comprisesgrowing the host vector system of the subject invention under conditionspermitting the production of the protein and recovering the proteinproduced thereby.

Methods and conditions for growing host vector systems and forrecovering the protein so produced are well known to those skilled inthe art, and may be varied or optimized depending upon the specificvector and host cell employed. Such recovery methods include, by way ofexample, gel electrophoresis, ion exchange chromatography, affinitychromatography or combinations thereof.

The subject invention further provides a method of determining whetheran agent is capable of specifically inhibiting the ability of p27protein to inhibit the activation of cyclin E-Cdk2 complex whichcomprises: (a) contacting suitable amounts of p27 protein, cyclin E,Cdk2 and the agent under suitable conditions; (b) subjecting the p27,cyclin E, Cdk2, and agent so contacted to conditions which would permitthe formation of active cyclin E-Cdk2 complex in the absence of p27protein; (c) quantitatively determining the amount of active cyclinE-Cdk2 complex so formed; and (d) comparing the amount of active cyclinE-Cdk2 complex so formed with the amount of active cyclin E-Cdk2 complexformed in the absence of the agent, a greater amount of active cyclinE-Cdk2 complex formed in the presence of the agent than in the absenceof the agent indicating that the agent is capable of specificallyinhibiting the ability of p27 protein to inhibit the activation ofcyclin E-Cdk2 complex.

As used herein, the term “agent” includes both protein and non-proteinmoieties. In one embodiment, the agent is a small molecule. In anotherembodiment, the agent is a protein. The agent may be derived from alibrary of low molecular weight compounds or a library of extracts fromplants or other organisms.

In the subject invention, an agent capable of specifically inhibitingthe ability of p27 protein to inhibit the activation of cyclin E-Cdk2complex interferes with the interaction between p27 protein and cyclinE-Cdk2 complex, but not with the site-specific phosphorylation of theCdk2 moiety of the cyclin E-Cdk2 complex in the absence of p27 protein.

Cyclin E may be obtained using methods well known to those skilled inthe art based on the nucleic acid sequence encoding same as disclosed inKoff, et al. (1991). Cdk2 may be obtained using methods well known tothose skilled in the art based on the nucleic acid sequence encodingsame as disclosed in Elledge and Spottswood (1991).

Amounts of p27 protein, cyclin E, Cdk2 and the agent suitable for themethod of the subject invention may be determined by methods well knownto those skilled in the art. An example of suitable conditions (i.e.,conditions suitable for measuring the effect on p27 function by anagent) under which p27 protein, cyclin E, Cdk2 and the agent arecontacted appears infra.

An example of conditions which would permit the formation of activecyclin E-Cdk2 complex in the absence of p27 protein also appears infra.

As used herein, “active cyclin E-Cdk2 complex” means a cyclin E-Cdk2complex which is capable of specifically phosphorylating a suitablesubstrate (e.g., histone H1). An example of an active cyclin E-Cdk2complex is provided infra. The amount of active cyclin E-Cdk2 complexcorrelates with its measurable activity. Thus, quantitativelydetermining the amount of active cyclin E-Cdk2 complex may beaccomplished by measuring the rate at which a substrate of the activecyclin E-Cdk2 complex is phosphorylated. Such methods well known tothose skilled in the art, and include, by way of example, a histone H1kinase assay.

In the method of the subject invention, the cyclin E and Cdk2 proteinsmay exist as separate proteins, or as a complex, prior to beingcontacted with the agent.

The subject invention further provides a method of determining whetheran agent is capable of specifically enhancing the ability of p27 proteinto inhibit the activation of cyclin E-Cdk2 complex which comprises: (a)contacting suitable amounts of p27 protein, cyclin E, Cdk2 and the agentunder suitable conditions; (b) subjecting the p27 protein, cyclin E,Cdk2, and agent so contacted to conditions which would permit theformation of active cyclin E-Cdk2 complex in the absence of p27 protein;(c) quantitatively determining the amount of active cyclin E-Cdk2complex so formed; and (d) comparing the amount of active cyclin E-Cdk2complex so formed with the amount of active cyclin E-Cdk2 complex formedin the absence of the agent, a lesser amount of active cyclin E-Cdk2complex formed in the presence of the agent than in the absence of theagent indicating that the agent is capable of specifically enhancing theability of p27 protein to inhibit the activation of cyclin E-Cdk2complex.

In the subject invention, an agent capable of specifically enhancing theability of p27 protein to inhibit the activation of cyclin E-Cdk2complex affects the interaction between p27 protein and cyclin E-Cdk2complex, but not with the site-specific phosphorylation of the Cdk2moiety of the cyclin E-Cdk2 complex in the absence of p27 protein.

The subject invention further provides a method of determining whetheran agent is capable of mimicking the ability of p27 protein to inhibitthe activation of cyclin E-Cdk2 complex which comprises: (a) contactingsuitable amounts of cyclin E, Cdk2 and the agent under conditions whichwould permit the formation of active cyclin E-Cdk2 complex in theabsence of the agent; (b) quantitatively determining the amount ofactive cyclin E-Cdk2 complex so formed; and (c) comparing the amount ofactive cyclin E-Cdk2 complex so formed with the amount of active cyclinE-Cdk2 complex formed in the absence of the agent, a lesser amount ofactive cyclin E-Cdk2 complex formed in the presence of the agent than inthe absence of the agent indicating that the agent is capable ofmimicking the ability of p27 protein to inhibit the activation of cyclinE-Cdk2 complex.

The subject invention further provides a method of treating a subjecthaving a hyperproliferative disorder which comprises administering tothe subject a therapeutically effective amount of an agent capable ofspecifically enhancing the ability of p27 protein to inhibit theactivation of cyclin E-Cdk2 complex in the hyperprolifera-tive cells ofthe subject, so as to thereby treat the subject.

In the preferred embodiment, the subject is a human.

A hyperproliferative disorder is a disorder wherein cells present in thesubject having the disorder proliferate at an abnormally high rate,which abnormally high rate of proliferation is a cause of the disorder.In one embodiment, the hyperproliferative disorder is selected from thegroup consisting of cancer and hyperplasia.

The administering of the agent may be effected or performed using any ofthe various methods known to those skilled in the art. In oneembodiment, the administering comprises administering intravenously. Inanother embodiment, the administering comprises administeringintramuscularly. In yet another embodiment, the administering comprisesadministering subcutaneously.

The therapeutically effective amount of the agent may be determined bymethods well known to those skilled in the art.

The subject invention further provides a pharmaceutical compositioncomprising a therapeutically effective amount of an agent capable ofspecifically enhancing the ability of p27 protein to inhibit theactivation of cyclin E-Cdk2 complex in the hyperproliferative cells of asubject suffering from a hyperproliferative disorder, and apharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are well known to those skilled inthe art and include, but are not limited to, 0.01-0.1M and preferably0.05M phosphate buffer or 0.8% saline. Additionally, suchpharmaceutically acceptable carriers may be aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers such as thosebased on Ringer's dextrose, and the like. Preservatives and otheradditives may also be present, such as, for example, antimicrobials,antioxidants, chelating agents, inert gases and the like.

The subject invention further provides a method of treating a subjecthaving a hyperproliferative disorder which comprises administering tothe subject a therapeutically effective amount of an agent capable ofmimicking the ability of p27 protein to inhibit the activation of cyclinE-Cdk2 complex in the hyperproliferative cells of the subject, so as tothereby treat the subject.

The subject invention further provides a method of treating a subjecthaving a hypoproliferative disorder which comprises administering to thesubject a therapeutically effective amount of an agent capable ofspecifically inhibiting the ability of p27 protein to inhibit theactivation of cyclin E-Cdk2 complex in the hypoproliferative cells ofthe subject, so as to thereby treat the subject.

In the preferred embodiment, the subject is a human.

A hypoproliferative disorder is a disorder wherein cells present in thesubject having the disorder proliferate at an abnormally low rate, whichabnormally low rate of proliferation is a cause of the disorder. In oneembodiment, the hypoproliferative disorder is an ulcer. Examples ofhypoproliferative cells are terminally differentiated cells in normaltissue and organs which, with the exception of the liver and bonemarrow, normally lack the ability to regenerate following traumaticinjury. Thus, the method of the subject invention, and agents identifiedthereby, have use in stimulating tissue and organ repair in subjects inneed thereof, as well as in establishing tissue cultures of cells from avariety of different tissues.

The therapeutically effective amount of the agent may be determined bymethods well known to those skilled in the art.

The subject invention further provides a pharmaceutical compositioncomprising a therapeutically effective amount of an agent capable ofspecifically inhibiting the ability of p27 protein to inhibit theactivation of cyclin E-Cdk2 complex in the hypoproliferative cells of asubject suffering from a hypoproliferative disorder, and apharmaceutically acceptable carrier.

The subject invention further provides a method for obtaining partiallypurified polyclonal antibodies capable of specifically binding to p27protein which method comprises (a) immunizing a subject with p27protein, (b) recovering from the immunized subject serum comprisingantibodies capable of specifically binding to p27 protein, and (c)partially purifying the antibodies present in the serum, therebyobtaining partially purified polyclonal antibodies capable ofspecifically binding to p27 protein.

As used herein, partially purified antibodies means a composition whichcomprises antibodies which specifically bind to p27 protein, andconsists of fewer protein impurities than does the serum from which theantibodies are derived. A protein impurity means a protein other thanthe antibodies specific for p27 protein. For example, the partiallypurified antibodies might be an IgG preparation.

Methods of recovering serum from a subject are well known to thoseskilled in the art. Methods of partially purifying antibodies are alsowell known to those skilled in the art, and include, by way of example,filtration, ion exchange chromatography, and precipitation.

The subject invention further provides the partially purified antibodiesproduced by the method of the subject invention.

The subject invention further provides a method for obtaining a purifiedmonoclonal antibody capable of specifically binding to p27 protein whichmethod comprises (a) immunizing a subject with p27 protein, (b)recovering from the immunized subject a B cell-containing cell sample,(c) contacting the B cell-containing cell sample so recovered withmyeloma cells under conditions permitting fusion of the myeloma cellswith the B cells therein so as to form hybridoma cells, (d) isolatingfrom the resulting sample a hybridoma cell capable of producing amonoclonal antibody capable of specifically binding to p27 protein, (e)growing the hybridoma cell so isolated under conditions permitting theproduction of the monoclonal antibody, and (f) recovering the monoclonalantibody so produced, thereby obtaining a purified monoclonal antibodycapable of specifically binding to p27 protein. Methods of makinghybridomas and monoclonal antibodies are well known to those skilled inthe art.

The subject invention further provides the hybridoma cell produced instep (d) of the method of the subject invention.

The subject invention further provides the purified monoclonal antibodyproduced by the method of the subject invention.

As used herein, a “purified monoclonal antibody” means the monoclonalantibody free of any other antibodies.

The subject invention further provides an antibody capable ofspecifically binding to p27 protein, said antibody being labeled with adetectable marker.

The labeled antibody may be a polyclonal or monoclonal antibody. In oneembodiment, the labeled antibody is a purified labeled antibody. Theterm “antibody” includes, by way of example, both naturally occurringand non-naturally occurring antibodies. Specifically, the term“antibody” includes polyclonal and monoclonal antibodies, and fragmentsthereof. Furthermore, the term “antibody” includes chimeric antibodiesand wholly synthetic antibodies, and fragments thereof. The detectablemarker may be, for example, radioactive or fluorescent. Methods oflabeling antibodies are well known in the art.

The subject invention further provides a method for quantitativelydetermining the amount of p27 protein in a sample which comprisescontacting the sample with the antibody of the subject invention underconditions permitting the antibody to form a complex with p27 proteinpresent in the sample, quantitatively determining the amount of complexso formed, and comparing the amount so determined with a known standard,so as to thereby quantitatively determine the amount of p27 protein inthe sample.

The sample may be, for example, a cell sample, tissue sample, orprotein-containing fluid sample. Conditions permitting an antibody toform a complex with its antigen and methods of detecting the presence ofcomplex so formed are well known in the art.

The amount of p27 protein present in a sample as determined need not bean absolute number, in the sense that it need not be the actual numberof p27 protein molecules or moles of p27 protein in the sample. Rather,the amount determined may merely correlate with this number.

The subject invention further provides a method for quantitativelydetermining the level of expression of p27 in a cell population, and amethod for determining whether an agent is capable of increasing ordecreasing the level of expression of p27 in a cell population. Themethod for determining whether an agent is capable of increasing ordecreasing the level of expression of p27 in a cell population comprisesthe steps of (a) preparing cell extracts from control and agent-treatedcell populations, (b) isolating p27 from the cell extracts (e.g., byaffinity chromatography on, and elution from, a cyclin E-Cdk2 complexsolid phase affinity adsorbant), (c) quantifying (e.g., in parallel) theamount of p27 inhibitor activity in the control and agent-treated cellextracts using a cyclin E-Cdk2 kinase assay (e.g., histone H1 assaydescribed infra). Agents that induce increased p27 expression may beidentified by their ability to increase the amount of p27 inhibitoractivity in the treated cell extract in a manner that is dependant ontranscription, i.e., the increase in p27 inhibitor activity is preventedwhen cells are also treated with an inhibitor of transcription (e.g.,actinomycin D). In a similar manner, agents that decrease expression ofp27 may be identified by their ability to decrease the amount of p27inhibitor activity in the treated cell extract in a manner that isdependent upon transcription.

The subject invention further provides a method of determining whether acell sample obtained from a subject possesses an abnormal amount of p27protein which comprises (a) obtaining a cell sample from the subject,(b) quantitatively determining the amount of p27 protein in the sampleso obtained, and (c) comparing the amount of p27 protein so determinedwith a known standard, so as to thereby determine whether the cellsample obtained from the subject possesses an abnormal amount of p27protein.

The subject invention further provides a method of determining whetherthe amount of p27 protein in a cell sample obtained from a subjecthaving a disease is correlative with the disease which comprisesdetermining whether a cell sample obtained from the subject possesses anabnormal amount of p27 protein, an abnormal amount of p27 protein in thesample indicating that the amount of p27 protein in the cell sampleobtained from the subject having the disease is correlative with thedisease.

The subject invention further provides a method of quantitativelydetermining the specific activity of p27 protein in a sample whichcomprises quantitatively determining (i) the ability of the p27 proteinin the sample to inhibit the activation of cyclin E-Cdk2 complex and(ii) the total amount of p27 protein in the sample, and dividing theability of the p27 protein so determined by the total amount of p27protein so determined so as to thereby quantitatively determine thespecific activity of p27 protein in the sample.

The subject invention further provides a kit for practicing the methodsof the subject invention. In one embodiment, the kit comprises suitableamounts of p27 protein, cyclin E and Cdk2. The kit may further comprisesuitable buffers, and a package insert describing p27 as an inhibitor ofcyclin E-Cdk2 complex activity.

The subject invention further provides a method of diagnosing ahyperproliferative disorder in a subject which disorder is associatedwith the presence of a p27 protein mutation in the cells of the subject,which comprises determining the presence of a p27 protein mutation inthe cells of the subject, said mutation being associated with ahyperproliferative disorder, so as to thereby diagnose ahyperproliferative disorder in the subject.

As used herein, “diagnosing” means determining the presence of ahyperproliferative disorder in a subject. In one embodiment,“diagnosing” additionally means determining the type ofhyperproliferative disorder in a subject.

As used herein, a “p27 protein mutation” may be any abnormality in theprimary sequence of p27 protein resulting from an abnormality in thegenomic DNA sequence encoding same or controlling the expression ofsame. For example, the p27 protein mutation may be a point mutation, adeletion mutation of a portion of p27 protein, or an absence of theentire p27 protein resulting from an abnormality in the structural geneencoding same or regulatory DNA sequence controlling the expression ofsame.

Determining the presence of a p27 protein mutation may be accomplishedaccording to methods well known to those skilled in the art. Suchmethods include probing a subject's DNA or RNA with a p27 nucleic acidprobe. Such methods also include analyzing a protein sample from thesubject for p27 protein structural abnormalities or functionalabnormalities resulting therefrom.

In the preferred embodiment, the subject is a human and thehyperproliferative disorder is cancer.

The subject invention further provides a pharmaceutical compositionwhich comprises an effective amount of a recombinant virus capable ofinfecting a suitable host cell, said recombinant virus comprising thenucleic acid molecule of the subject invention, and a pharmaceuticallyacceptable carrier.

The “suitable host cell” is any cell in which p27 protein would normallybe produced in a healthy subject.

Pharmaceutically acceptable carriers are well known to those skilled inthe art and include, but are not limited to, 0.01-0.1M and preferably0.05M phosphate buffer or 0.8% saline. Additionally, suchpharmaceutically acceptable carriers may be aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers such as thosebased on Ringer's dextrose, and the like. Preservatives and otheradditives may also be present, such as, for example, antimicrobials,antioxidants, chelating agents, inert gases and the like.

Finally, this invention provides a method for treating a subjectsuffering from a hyperproliferative disorder associated with thepresence of a p27 protein mutation in the cells of the subject, whichcomprises administering to the subject an amount of the pharmaceuticalcomposition of the subject invention effective to treat the subject.

In the preferred embodiment, the subject is a human and thehyperproliferative disorder is cancer.

In order to facilitate an understanding of the Experimental Detailssection which follows, certain frequently occurring methods and/or termsare best described in Sambrook, et al. (1989).

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS I Summary

Cell-cell contact and TGF-β can arrest the cell cycle in G1. Mv1Lu minkepithelial cells arrested by either mechanism are incapable ofassembling active complexes containing the G1 cyclin, cyclin E, and itscatalytic subunit, Cdk2. These growth inhibitory signals block Cdk2activation by raising the threshold level of cyclin E necessary toactivate Cdk2. In arrested cells the threshold is set higher thanphysiological cyclin E levels, and is determined by an inhibitor thatbinds to cyclin E-Cdk2 complexes. A 27 kD protein that binds to andprevents the activation of cyclin E-Cdk2 complexes can be purified fromarrested cells, but not from proliferating cells, using cyclin E-Cdk2affinity chromatography. p27 is present in proliferating cells, but itis sequestered and unavailable to interact with cyclin E-Cdk2 complexes.Cyclin D2-Cdk4 complexes competitively bind to and down-regulate theactivity of p27 and may thereby act in a pathway that reverses Cdk2inhibition and enables G1 progression.

Methods

Cell Culture

Exponentially growing Mv1Lu cells were growth arrested by culturing themto confluence in the presence of 10% fetal bovine serum. Cells werereleased from contact inhibition by trypsinization and reseeding insparse conditions. TGF-β (100 pM) was added to the cells at theindicated times. Cell entry into S phase was routinely confirmed bymeasuring ¹²⁵Ideoxyuridine incorporation into DNA (Laiho et al., 1990).

Preparation of Recombinant Proteins

Cyclin E, Cdk2, Cdk2-HA, and Cdk2K were prepared by the method of Desaiet al (1992). Briefly, 100 mm plates of confluent Sf9 cells wereinfected with the appropriate baculovirus at an m.o.i. of 5-20 p.f.u percell. After 48 hours of infection the cells were collected and lysed byDounce homogenization or cup-horn sonication in hypotonic buffer. Theextract is clarified by ultracentrifugation and stored at −70° C. Thebaculoviral vectors containing cyclins D1, D2, D3, Cdk4 andcatalytically inactive Cdk4 have been previously described (Matsushimeet al., 1992: Kato et al., 1993).

Purification and Microsequencing of p27^(Kip-1)

In order to purify p27 in amounts sufficient for microsequence analysis,the purification protocol used as the starting material 200 15-cm dishescontaining confluent cultures of contact inhibited Mv1Lu cells (˜2×10¹⁰cells). Cells were lysed by sonication in 33 ml of hypotonic extractionbuffer, and cell debris were removed by centrifugation at 200,000×g for1 hour. The lysate was heated to 100° C. for 5 min, and precipitatedmaterial was removed by centrifugation at 100,000×g for 15 min. Thesupernatant was adjusted to NP-40 lysis buffer conditions (Polyak etal., 1994) with 4× NP-40 lysis buffer, and precleared by two successive30 min. incubations with 5 ml of agarose at 4° C. and once with 5 ml ofnickel-NTA-agarose under the same conditions.

The precleared lysate was allowed to bind to an affinity column for 2hours at 4° C. This affinity column consisted of nickel-NTA-agarosecontaining baculoviral Cdk2 in complex with baculoviral cyclin E taggedat the N-terminus with a hexahistidine sequence that allows binding tonickel-NTA-agarose. The column was washed once with 50 ml of NP-40 lysisbuffer, and five times with 50 ml of SDS/RIPA buffer at roomtemperature. Bound proteins were eluted with 5 ml of a Hepes-bufferedsolution (pH 7.0) containing 6M guanidium hydrochloride. The eluate wasdialyzed overnight against Hepes-buffered solution, and proteins wereprecipi-tated with 4 volumes of acetone at −20° C. for 30 min.Precipitated proteins were collected by centrifugation at 20,000×g for15 min, solubilized in SDS electrophoresis sample buffer containingdithiothreitol, and electrophoresed on a 12% polyacrylamide gel. Afterelectrophoresis, the gel was blotted onto nitrocellulose at 35Vovernight in Tris/glycine/methanol transfer buffer.

The nitrocellulose membrane was stained with Ponceau stain to detectproteins. According to this assay, the filters contained only twoproteins that were well separated from each other and were of 27 kd and34 kd, respectively. These proteins were identified as p27 andp34^(cdk2). Two separate preparations gave similar results. The yield ofp27 in these two preparation was approximately 0.3 μg and 1 μg,respectively, as estimated by from the Ponceau staining and frommicrosequencing.

The protein of nitrocellulose containing purified p27 was excised andsubjected to tryptic digestion in preparation for microsequencinganalysis. After HPLC of the tryptic digests, the following peptides weresequenced:

1. Asn-Leu-Tyr-Pro-Leu-Thr-Asn-Tyr-Thr-Phe (SEQ ID NO: 7)

2. Thr-Asp-Thr-Ala-Asp-Asn-Gln-Ala-Gly-Leu-Ala-Glu-Gln (SEQ ID NO: 8)

3. Gln-Ala-Val-Pro-Leu-Met-Gly-Pro-Gln-Glu (SEQ ID NO: 9)

4. Leu-Pro-Glu-Phe-Tyr-Tyr-Arg-Pro-Pro-Arg-Pro-Pro (SEQ ID NO: 10)

5. Tyr-Glu-Trp-Gln-Glu-Val (SEQ ID NO: 11)

No similarity has been found between these sequences and proteinsequences deposited in Genbak, EMBL Data Library, Brookhaven ProteinDatabank, Swiss Prot or PIR databases, according to the updatesavailable in Dec. 31, 1993.

Oligonucleotides for Obtaining p27 cDNA

Oligonucleotides to be used in obtaining the full-length cDNA sequenceof p27 are shown in Table II:

TABLE II Peptide #1: None Peptide #2: Sense5′-AC(N)-GA(T/C)-AC(N)-GA(T/C)-AA(T/C)- (SEQ ID NO: 12) CA(A/G)-GC-3′Antisense 5′-(N)GC-(T/C)TG-(A/G)TT-(A/G)TC-(N)GC- (SEQ ID NO: 13)(N)GT-(A/G)TC-(N)GT-3′ Peptide #3: Sense5′-CA(A/G)-GC(N)-GT(N)-CC(N)-CT(N)-ATG-GG- (SEQ ID NO: 14) 3′ and5′-CA(A/G)-GC(N)-GT(N)-CC(N)-TT(A/G)-ATG (SEQ ID NO: 15) -GG-3′Antisense 5′-(N)CC-CAT-(N)AG-(N)GG-(N)AC-(N)GC- (SEQ ID NO: 16)(T/C)TG-3′ and 5′-(N)CC-CAT-(T/C)AA-(N)GG-(N)AC-(N)GC- (SEQ ID NO: 17)(T/C)TG-3′ Peptide #4: Sense 5′-CC(N)-GA(A/G)-TT(T/C)-TA(T/C)-TA(T/C)-(SEQ ID NO: 18) (C/A)G-3′ Antisense5′-C(T/G)-(A/G)TA-(A/G)TA-(A/G)AA-(T/C)TC- (SEQ ID NO: 19) (N)GG-3′Peptide #5: Sense 5′-TA(T/C)-GA(A/G)-TGG-CA(A/G)-GA(A/G)-GT- (SEQ ID NO:20) 3′ Antisense 5′-(N)AC-(T/C)TC-(T/C)TG-CCA-(T/C)TC- (SEQ ID NO: 21)(A/G)TA-3′

CAK

CAK was purified from Xenopus egg extracts through the Mono Q stepexactly as described (Solomon et al., 1993) and was used at a finalconcentration of 1-2 units per ml.

Metabolic Labeling

Mv1Lu cultures in 150 mm dishes were incubated for 30 minutes inmethionine-free medium supplemented with 10% dialyzed fetal bovineserum, followed by incubation for 2 hours in the same medium with 200μCi/ml of 35S-methionine (Trans 35S label, ICN). Cells were collected bytrypsini-zation and centrifuged at 2000 g for 5 minutes. Cell pelletswere lysed by gentle agitation for 30 minutes at 4° C. in 10 volumes ofNP40 lysis buffer (50 mM Tris HCl pH 7.4, 200 mM NaCl, 2 mM EDTA, 0.5%NP40, 0.3 mM Na-orthovanadate, 50 mM NaF, 80 μM b-glycerophosphate, 20mM Na pyrophosphate, 0.5 mM DTT and protease inhibitors) and lysateswere clarified by centrifugation (10,000 g 15 minutes at 4° C.). Priorto binding reactions the supernatants were precleared twice withsepharose and once with protein A-sepharose.

Cdk Activation Assays

Indicated amounts of baculovirus expressed recombinant cyclin, Cdk, orcyclin-Cdk complex were added to 50 micrograms of extracts prepared bysonication in a hypotonic buffer as previously described (Koff et al1993). In all cases the exogenous cyclins and Cdks were added in theform of an unfractionated Sf9 cell lysate. Cyclins and Cdks typicallycomprise at least 1-3% of total cell protein. Uninfected Sf9 celllysates have been tested in all assays and have no activity. After 30minutes at 37° C. the reaction was adjusted to 0.5% NP40, 250 mM NaCland immunopre-cipitated with the indicated antibody. Immunoprecipitateswere subsequently assayed for histone H1 kinase activity as described(Koff et al 1993). For experiments in which the effect of the D cyclinsand Cdk4 on cyclin E activity were tested, all cyclins and Cdks wereadded to the cell extract together. Heat treatment of extracts wasperformed by incubating extracts to 100° C. for 5 minutes. Coagulatedprotein was then pelleted by microcentrifugation. For experiments inwhich Cdk2 immunoprecipitates were tested for activation by cyclin E, 20μl of antiserum to the C-terminus of CDK2 (Koff et al, 1993) wasadsorbed to protein A sepharose and washed into NP40 RIPA buffer. 300 μgof extract was subsequently incubated with the anti-CDK2 sepharose for90 minutes at 4° C. The precipitate was washed twice with NP40 RIPAbuffer and 4 times with buffer A containing 10 mM ATP. Cyclin E and CAKwere added as described below and reactions were incubated for 30minutes at 37° C. and subsequently assayed for H1 kinase activity.

Inhibitor Depletion

Cyclin E-Cdk2 sepharose was prepared by mixing 1.2 μl of Sf9 cell lysatecontaining Hemagglutinin tagged Cdk2 (Cdk2 -HA) with 12 μl lysatecontaining cyclin E in buffer A (30 mM HEPES-KCH pH 7.5, 7.5 mM MgCl₂, 1mM DTT) containing 10 mM ATP and incubated at room temperature for 30minutes to allow complete formation. The assembly reaction was thenadjusted to 250 mM NaCl and 0.5% NP40. The Cdk2-HA containing complexeswere immunoprecipitated with the 12CA5 monoclonal antibody (BABCO) andcollected on protein A-sepharose. Cdk2 sepharose was prepared in anidentical manner except cyclin E was omitted. Immunoprecipitates werewashed twice with NP40 RIPA buffer (0.5-0 NP40, 250 mM NaCl, 10 mM EDTA,20 mM Tris-HCl pH 7.4) and four times with buffer A. The matrix wasdivided into 4 aliquots and incubated with 100 μg of cell extract inbuffer A containing 3 mM ATP, 20 μg/ml creatine phosphokinase, 40 mMphospho-creatine for 45 minutes at 37° C. After incubation thesupernatant was collected and assayed for Cdk2 activation by addition ofrecombinant cyclin E as described below. A critical parameter in theexecution of this experiment is to ensure that no cyclin, Cdk or complexleaks from the beads into the cell extract. This is unpredictable andmust be checked by immunoblotting each time the experiment is performed.

Cyclin E-Cdk2 Bindinq Assays

Complexes of baculoviral cyclin E with baculoviral Cdk2 containing theinfluenza virus Hemagglutinin epitope HA1 were formed as describedbelow. The complexes were immunoprecipitated in NP40-RIPA buffer (50 mMTris-HCl pH=7.4, 250 mM NaCl, 0.5% NP-40, 50 mM NaF, 0.3 mMNa-ortho-vanadate, 5 mM EDTA and protease inhibitors) with anti-HAmonoclonal antibody (12CA5, BabCo) and bound to protein A sepharose.Cdk2 or cyclin E-Cdk2 adsorbed to protein A-sepharose were incubatedwith metabolically labeled cell lysates from 10⁷ cells for 2 h at 4° C.Unless otherwise indicated, the beads were washed several times withSDS-RIPA buffer, and the proteins were eluted by heating in SDS-PAGEsample buffer and analyzed on 12% polyacrylamide gels followed byfluorography. For heat treatment metabolically labeled cell lysates wereheated for 3 minutes at 100° C., the precipitated proteins were removedby microcentrifu-gation and the clarified lysates were incubated withprotein A-Sepharose bound Cdk2 or cyclin ECdk2 complexes. In bindingassays using cyclin D2-Cdk4 complexes, metabolically labeled cellextracts were pre-incubated with 4 μl of cyclin D2-Cdk4 complex for 30minutes at 4° C. before addition of protein A-sepharose bound Cdk2 orcyclin E-Cdk2. After removing the sepharose beads, cell extracts wereimmunoprecipitated with Cdk4 antiserum and the immunoprecipitates wereanalyzed on 120 SDS-PAGE.

Affinity Purification of p27 and Denaturation-renaturation Experiments

HA-tagged Cdk2, alone or in complex with cyclin E, was bound to HAantibody immobilized on protein A sepharose beads (ImmunoPureOrientation Kit, Pierce,) and used to isolated proteins frommetabolically labeled cell lysates. Bound proteins were eluted from thecolumn in 0.1M glycine pH 2.8 and precipitated with 4 volumes ofice-cold acetone and kept at −20° C. for 20 minutes. The precipitatescollected by microcentrifugation for 30 minutes were washed severaltimes with cold acetone, and dissolved in 6M guanidium chloride in 1×HBBbuffer (25 mM HEPES-KOH pH 7.7, 25 mM NaCl, 5 mM MgCl₂, 0.05% NP-40, 1mM DTT). For renaturation (Kaelin et al., 1992), samples were dialyzedovernight against 1×HBB buffer and used either in kinase inhibitionassays or for binding to cyclin E-Cdk2 sepharose. For cyclin Eassociated H1 kinase inhibition assays, aliquots (37.5 llg of protein)from 100,000×g supernatants of lysates prepared from exponentiallygrowing Mv1Lu cells, were incubated for 30 min. at 37° C. withphysiological amounts of baculoviral cyclin E either alone or in thepresence of the indicated volumes of renatured eluates. Afterincubation, samples were precipitated with cyclin E antiserum, andassayed for histone H1 kinase activity. The relative cyclin E-associatedH1 kinase activity was quantitated using a Molecular DynamicsPhosphorimager ImageQuant software.

To assay the activity of protein eluted from gel slices cyclin E Cdk2-HAaffinity column eluates were run on 12% polyacrylamide gels along withmolecular weight markers (Amersham). Part of the sample was run on thesame gel, stained with Commassie, destained and detected byfluorography. The gel was cut as indicated (between 0.5 to 1 cm/slice)and the proteins were isolated from the gel as described (Boyle et al.,1991). The isolated proteins were renatured and used for kinaseinhibition assays as described below.

Results

Non-proliferating Cells Contain an Inhibitor of Cdk2 Activation

Cell free extracts from contact inhibited, TGF-β arrested andproliferating cells were used to investigate the mechanism that blocksactivation of the cyclin E-Cdk2 complex. It has been shown that additionof physiological amounts of cyclin E to these cell extracts resulted inan increase in the amount of immunoprecipitable cyclin E-Cdk2 complexes;however, only the cyclin E-Cdk2 complexes assembled in extracts fromproliferating cells were enzymatically active using histone H1 as asubstrate (Koff et al, 1993; see also FIG. 1A). Cell extracts,therefore, recapitulate the block to Cdk2 activation observed in intactcells.

The block to Cdk2 activation in extracts from non-proliferating cellscould be overcome by addition of cyclin E protein to greater thanphysiological levels (FIG. 1A). Cyclin E was expressed in Sf9 cellsusing a baculoviral expression vector and the amount of cyclin E in Sf9extracts was compared to that in Mv1Lu cell extracts by immuno-blotting(not shown). In the experiment illustrated in FIG. 1A, 0.05 μl of Sf9lysate contained as much cyclin E as 50 μg of total cell protein fromMv1Lu cell lysates. Addition of cyclin E (in the form of Sf9 lysate) toan extract from proliferating cells gave a linear increase in cyclinE-associated histone H1 kinase activity (prolifera-ting cells wereharvested 15 hours after release from contact inhibition, at which timethey were in early S phase). In contrast, titration of up to 3 timesphysio-logical levels of cyclin E into extracts from contact inhibitedor TGF-β treated cells resulted in no increase in immunoprecipitablecyclin E-associated kinase activity. As more cyclin E was added, cyclinE-associated kinase activity became detectable and increased inproportion. Thus, extracts from non-proliferating cells demonstrated anelevated threshold level of cyclin E necessary to activate Cdk2. Contactinhibited cells appeared to have a higher threshold than cells arrestedin G1 by exposure to TGF-β, but in both cases the cyclin E requirementwas substantially greater than the physiological levels of cyclin Eachieved in proliferating cells.

Supra-physiological amounts of cyclin E were required to activate Cdk2in extracts from non-proliferating cells. This could not be explained bylower levels of Cdk2 or cyclin E, nor did these cells appear to lackother factors necessary for Cdk2 activation (Koff et al., 1993; seebelow). One explanation was that non-proliferating cells contained atitratable inhibitor of Cdk2 activation. Mixing experiments supportedthis conclusion. Extracts from proliferating cells were mixed with thosefrom either contact inhibited or TGF-β treated cells. Physiologicallevels of cyclin E were added to the mixed extracts and then cyclin Eand any associated kinases were immunoprecipitated using antibodies tothe cyclin. Identical results were obtained using an anti-Cdk2 antiserum(not shown). In mixed extracts cyclin E-associated kinase activity wasreduced below that recovered from extracts of proliferating cells alone(FIG. 1B). Thus, extracts from non-proliferating cells contained anexcess of an inhibitor of Cdk2 activation. Note that extracts fromcontact inhibited cells had both a higher cyclin E activation thresholdand a greater inhibitory effect in mixing experiments than extracts fromTGF-β treated cells. However, the abundance of the Cdk2 inhibitoryactivity depended upon the duration of exposure to TGF-β. For instance,it is shown that an extract from cells exposed to TGF-β for 6 hoursbeginning in late G1 did not contain sufficient inhibitory activity toblock Cdk2 activation when mixed with an extract from proliferatingcells (Koff et al., 1993) and cells exposed to TGF-β for 48 hours hadmore inhibitory activity that cells exposed for 15 hours (not shown).

A Cdk2 Inhibitor Binds to Cyclin E-Cdk2 Complexes

The inhibitor of Cdk2 activation present in extracts fromnon-proliferating cells could be depleted using a cyclin E-Cdk2 affinitymatrix. Cyclin E-Cdk2 complexes were formed by mixing extracts from Sf9cells infected with baculoviral vectors expressing either Cdk2 taggedwith an influenza virus hemagglutinin (HA) epitope or cyclin E. Althoughneither extract alone contains significant H1 kinase activity, mixing ofthe extracts yields high levels of active enzyme (Kato et al., 1993).The cyclin E-Cdk2 (HA) complexes were immunoprecipitated withsepharose-linked monoclonal antibody directed against the HA tag onCdk2. Control immunoprecipitations were performed using the monoclonalantibody beads alone. Cell extracts were incubated with either thecyclin E-Cdk2 beads or the control beads, and after pelleting thesupernatants were assayed for the ability of exogenously added cyclin Eto activate endogenous Cdk2. After depletion of cyclin E-Cdk2 bindingproteins, cyclin E was able to activate Cdk2 almost equally in extractsfrom proliferating and non-proliferating cells (FIG. 2A). Immunoblottingshowed that this protocol had no effect on the levels of either cyclin Eor Cdk2 in the cell extracts (not shown). In this experiment somestimulatory effect of depleting cyclin E-Cdk2 binding proteins was alsoobserved in extracts from proliferating late G1 cells, suggesting theyare not completely devoid of the inhibitor (see below). Complexescontaining cyclin E and a catalytically inactive mutant of Cdk2 alsowere able to sequester inhibitory activity when added directly to cellextracts (see FIG. 2C). Thus reversal of inhibitory activity did notrequire phosphorylation by the added cyclin E-Cdk2 complexes. Theseexperiments showed that the inhibitor of Cdk2 activation bound to cyclinE-Cdk2 complexes.

In parallel it is observed that beads containing just Cdk2 alone wereunable to deplete the inhibitory activity from cell extracts (FIG. 2A).This experiment suggested that the inhibitor bound to cyclin E-Cdk2complexes but not to Cdk2 alone. To directly test this idea, Cdk2 wasimmuno-precipitated from extracts of proliferating, contact inhibitedand TGF-β treated cells. In all cases, the immunoprecipitated Cdk2protein could be activated by addition of both cyclin E and p34cdc2Activating Kinase (CAK) (FIG. 2B). Thus, the Cdk2 protein innon-proliferating cells was not intrinsically incapable of activation,nor was it tightly associated with an inhibitor of activation.

Since the Cdk2 inhibitor could bind to cyclin E-Cdk2 complexes, but notto Cdk2, it appeared to recognize either the cyclin-Cdk complex orcyclin. Cyclin E-Cdk2 complexes were more effective at removing theinhibitory activity than was cyclin E, suggesting that the inhibitorinteracted preferentially with complexes. Cyclin E was added to anonproliferating cell extract at a level below the threshold necessaryto activate Cdk2 (FIG. 2C). The assembly of additional cyclin-Cdk2complexes was then induced by supplementing the extracts with anexogenous Cdk2 protein that was rendered catalytically inactive by amutation of its ATP binding site (Gu et al., 1992). In the absence ofextra Cdk2 no kinase activity was detected in cyclin Eimmunoprecipitates. When extracts were supplemented with catalyticallyinactive Cdk2, cyclin E regained H1 kinase activity as a result ofactivating the endogenous Cdk2. Thus, the cyclin E threshold for Cdk2activation could be lowered by assembling additional cyclin-Cdkcomplexes while keeping the total amount of cyclin E constant.

The Inhibitor is Neither an Anti-CAK nor a Tyrosine Kinase

Previous experiments (Koff et al., 1993) indicated that cyclin E-Cdk2complexes formed in extracts from non-proliferating cells were notphosphorylated at an essential threonine residue (Gu et al., 1992;Solomon et al., 1992) possibly accounting for their inactivity. Thisraised the possibility that CAK was a target of the inhibitor. Thisinitially seemed unlikely because the inhibitor bound directly to thecyclin E-Cdk2 complex. This idea was reconsidered in light of recentevidence that CAK is itself a distant member of the Cdk protein family(Fesquet et al., 1993; Poon et al., 1993; Solomon et al., 1993) andtherefore might also bind to the inhibitor. Previous work, in anothersystem, indicated that activation of the cyclin B-Cdc2 complex was notblocked by the Cdk2 inhibitor (see below). Cyclin B and Cdc2 weretherefore used to assay CAK activity, given that CAK is also required toactivate the cyclin B-Cdc2 complex (Solomon et al., 1990).

Cdc2 was activated equally when cyclin B was added to extracts fromeither proliferating cells or TGF-β arrested cells (FIG. 3A). Therefore,functional CAK was present in extracts from TGF-β treated cells. CAK waslimiting in this experiment since addition of purified CAK to theseextracts catalyzed the activation of additional cyclin B-Cdc2 complexes(FIG. 3B). Moreover, the activity of the added CAK was similar inextracts from TGF-β treated and proliferating cells (FIG. 3B). Thus,exogenous CAK was not inhibited. Control experiments showed that thisCAK was able to activate cyclin E-Cdk2 complexes when they wereassembled by mixing Sf9 cell lysates containing cyclin E and Cdk2expressed from baculoviral vectors (Solomon et al, 1993; data notshown). However, the added CAK did not change the threshold level ofcyclin E required to activate Cdk2 (not shown). Thus, the inhibitorneither blocked CAK nor could its effects be overcome by excess CAK.Inhibition of CAK was not sufficient to explain the block to Cdk2activation.

To determine if tyrosine phosphorylation contributed to the inhibitionof Cdk2 activity, cyclin E was added to non-proliferating cell extractsat sub-threshold levels and the cyclin E-Cdk2 complexes wereimmunoprecipitated using anti-cyclin E antibodies. No tyrosinephosphorylation of Cdk2 in the inactive cyclin E-Cdk2 complexes wasdetected by immunoblotting with anti-phosphotyrosine antibodies (notshown). As a positive control, phosphotyrosine was readily detected inCdc2 immunoprecipitated from human cells.

Cyclin D2-Cdk4 Complexes Facilitate Cdk2 Activation

As cells traverse G1, complexes between Cdk4 and the D-type cyclinsappear prior to the formation of active complexes containing cyclin Eand Cdk2 (reviewed in Sherr, 1993). Contact inhibited Mv1Lu cells do notexpress significant levels of cyclin D1 or D2 (not shown) and Cdk4synthesis is repressed in cells arrested in G1 by exposure to TGF-β(Ewen et al., 1993: K.P and J.M., unpublished observations; see alsoFIG. 5D). Thus, accumulation of cyclin D-Cdk4 complexes is limiting inG1 arrested cells. These obser-vations suggested that cyclin D-Cdk4complexes could potentially have a role in removing the Cdk2 inhibitorduring cell cycle progression. Indeed, Ewen et al.(1993b) recentlyshowed that constitutive ectopic expression of Cdk4 can override theTGF-β block to Cdk2 activation and cell cycle progression. Thisphenomenon was tested by asking whether the restoration of cyclin D-Cdk4complexes to extracts from non-proliferating cells might overcome theblock to Cdk2 activation.

Cdk4 is a partner of the D-type cyclins and does not form activecomplexes with cyclins E, A or B. It interacts equally well with each ofthe D-type cyclins when they are co-expressed in insect cells. CyclinD-Cdk4 complexes are poorly active on histone H1 but show strongactivity using the Rb protein as substrate (Matsushime et al., 1992;Kato et al., 1993). Complexes between Cdk4 and either cyclin D1, D2 orD3 were assembled by co-infection of Sf9 cells with baculoviral vectorsand Sf9 lysates were added to extracts from proliferating andnon-proliferating Mv1Lu cells. Sub-threshold amounts of cyclin E werethen added, and activation of Cdk2 was tested after immunoprecipitationof cyclin E-Cdk2 complexes with antibodies to cyclin E. Addition ofcyclin D2-Cdk4 complexes, but neither subunit alone, to extracts fromcontact inhibited and TGF-β arrested cells allowed cyclin E to activateCdk2 to a level equivalent to that observed in extracts fromproliferating cells (FIG. 4A). Titrations demonstrated that the amountof cyclin D2-Cdk4 necessary to block the Cdk2 inhibitor was less thanthat present in an equivalent amount of extract from proliferating cells(not shown). In contrast, the activity of cyclin E was not increasedwhen cyclin D2-Cdk4 complexes were added to extracts from proliferatingcells. Moreover, the cyclin D2-Cdk4 complex did not have CAK activity,since it was unable to substitute for CAK in promoting the activation ofcyclin E-Cdk2 complexes assembled from proteins expressed in Sf9 cells(not shown). Thus, the cyclin D2-Cdk4 complex reversed the inhibition ofCdk2 activation. Equal amounts of cyclin Di-Cdk4 and cyclin D3-Cdk4complexes, as estimated by immunoblotting of Sf9 lysates, were much lesseffective in lowering the cyclin E threshold for Cdk2 activation (FIG.4B). The inability of cyclin D1- or cyclin D3-Cdk4 complexes tosequester the Cdk2 inhibitor was not because those complexes wereunstable in cell lysates (not shown).

Quite surprisingly, the ability of cyclin D2-Cdk4 to reverse Cdk2inhibition did not require Cdk4 catalytic activity. Complexes formedbetween cyclin D2 and a catalytically inactive mutant Cdk4 subunit wereas effective as enzymatically active cyclin D2-Cdk4 complexes inremoving the Cdk2 inhibitor (FIG. 4A). Titrations with different amountsof cyclin D2 complexes containing either catalytically active orinactive Cdk4 revealed that their specific activities in reversing theCdk2 inhibition were very similar (not shown). This ruled out thepossibility that Cyclin D2-Cdk4 must phosphorylate the inhibitor toinactivate it, and excluded any model in which cyclin D2-Cdk4 bypassedthe inhibitor by functioning as a CAK. It, therefore, seemed likely thatcyclin D2-Cdk4 removed the Cdk2 inhibitor by binding to it directly andsequestering it from Cdk2 (see below).

The Cdk2 Inhibitor is a 27 kd Protein

The above observations indicated (i) that a functional cyclin E-Cdk2inhibitor was present in extracts from contact inhibited cells or cellsreleased from contact inhibition in the presence of TGF-β, but not inextracts from prolifera-ting cells; (ii) that this moleculepreferentially associated with cyclin E-Cdk2 complexes as opposed toeither subunit alone; and (iii) that it could be depleted bypreincubation of cell extracts with catalytically active or inactivecyclin D2-Cdk4 complexes. To identify a factor that might display theseproperties, Mv1Lu cells were metaboli-cally labeled with 35S-methionine,and lysates were incubated with Sepharose beads that containedimmunoadsorbed recombinant Cdk2, either alone or in complexes withrecombinant cyclin E. Denatured 35S-labeled proteins, eluted by heatingthe beads with buffer containing 1% SDS, were visualized by gelelectrophoresis and fluorography (FIG. 5A). All cell lysates yielded asimilar pattern of cyclin E-Cdk2—binding proteins with the exception ofa 27 kd protein that was recovered from extracts of contact-inhibited orTGF-β inhibited cells, but not late G1 phase cells (FIG. 5A). Thisprotein, referred to as p27, was isolated using cyclin E-Cdk2 complexesbut not Cdk2 alone (FIG. 5A). The recovery of p27 increased inproportion to the amount of cyclin ECdk2 complex used until it reached amaximum (FIG. 5B), indicating that binding of p27 to cyclin E-Cdk2complexes was saturable. This was consistent with the observation thatCdk2 inhibitor activity could be depleted by cyclin E-Cdk2 complexes. Asexpected, stoichio-metric amounts of p27 were also observed in cyclin Eimmunoprecipitates from growth arrested cells (not shown).

Cell extracts that received recombinant cyclin D2-Cdk4 complex no longeryielded p27 when the mixture was adsorbed to cyclin E-Cdk2-Sepharose(FIG. 5C). After removal of the cyclin E-Cdk2-Sepharose beads fromsamples that received cyclin D2-Cdk4, the precleared supernatants wereincubated with Cdk4 antibody to recover Cdk4 and its associatedproteins. This yielded p34Cdk4 itself, whose levels were highest inextracts from cells in late G1 and lowest in TGF-β treated cells (FIG.5D) (Matsushime et al., 1992; Ewen et al., 1993b). Using the sameantiserum, Ewen et al, (1993b) used partial proteolytic digestion toconfirm that this is authentic Mv1Lu Cdk4. In addition, theseimmuno-precipitates contained a 27 kD protein in samples fromcontact-inhibited and TGF-β treated cells (FIG. 5D). Lesser amounts ofp27 were also recovered in Cdk4 immuno-precipitates from late G1 cellsamples, even though p27 could not be recovered from those same extractsby cyclin E-Cdk2 affinity chromatography. This suggested that p27 waspresent in proliferating cells, but in a form unavailable to interactwith exogenously added cyclin E-Cdk2 complexes (see below). Side-by-sidecomparison showed that p27 purified on cyclin E-Cdk2 beads or byco-precipitation with Cdk4 had the same apparent molecular weight (notshown).

Experiments done to characterize the stability of this factor showedthat heating cell extracts to 100° C. for a brief period preserved boththe ability of p27 to bind to cyclin E-Cdk2 (FIG. 6A) and the inhibitoryactivity as well (FIG. 6C). Furthermore, when applied to extracts fromcells in late G1 phase, heat treatment unexpectedly induced theappearance of both p27 (FIG. 6A) and concomitantly increased the levelof Cdk2 inhibitory activity (FIG. 6B). These results indicated that p27and the inhibitory activity were both heat stable, and that they couldbe re-activated in late G1 extracts by a heat-sensitive mechanism. Asexpected, cyclin D2-Cdk4 complexes were also able to sequester p27 fromheat treated lysates (not shown).

Extracts from metabolically-labeled TGF-β treated cells were subjectedto chromatography over cyclin E-Cdk2-sepharose or, as a control,Cdk2-sepharose. After washing, the beads were eluted with an acidicbuffer, and one portion of the eluate was analyzed by SDS PAGE. Thisshowed that p27 was the predominant labeled species recovered and waspresent only in the eluate from cyclin E-Cdk2 beads (FIG. 7A). Samplesfrom the same eluates were assayed for the presence of the Cdk2inhibitor, and this activity was present in the eluate from cyclinE-Cdk2 beads but not Cdk2 beads (FIG. 7B). The remainder of the eluatewas concentrated by acetone precipitation, denatured in 6M guanidiumhydrochloride, renatured by dialysis against isotonic buffer andsubjected to a second round of binding to cyclin E-Cdk2 Sepharose.Elution from these beads by boiling in buffer containing 1% SDS yieldedp27 as the only labeled band (FIG. 7C). These results strongly supportedthe possibility that p27 and the inhibitory activity are one and thesame. This conclusion was directly confirmed by fractionating the cyclinE-Cdk2 eluate by polyacrylamide gel electrophoresis and extracting thefractionated proteins from gel slices. Renatured proteins were testedfor their ability to inhibit activation of Cdk2 by cyclin E (FIG. 7D).The protein recovered from the gel slice containing p27 completelyinhibited Cdk2 activation, and no additional inhibitory activity wasrecovered from any other gel slice.

Discussion

An inhibitor in non-proliferating cells was identified that preventsactivation of complexes containing the G1 cyclin, cyclin E (Koff et al.,1991; Lew et al., 1991; Ohtsubo & Roberts, 1993), and its catalyticsubunit, Cdk2 (Koff et al., 1992; Dulic et al., 1992). This inhibitoryactivity is, at least in part, attributable to a 27 kDa polypeptide,which has also been named p27^(Kip1) (Cdk inhibitory protein 1). Theinhibitor and p27^(Kip1) share the following characteristics: they bindto cyclin E-Cdk2 complexes but not to Cdk2 alone; they are only detectedin extracts from growth arrested cells; they can be sequestered bycyclin D2-Cdk4 complexes but not by either component alone; they areheat stable; they are latent in extracts of proliferating cells but canbe unmasked by brief heat treatment. In addition purified p27^(Kip1)inhibits Cdk2 activation by cyclin E when added to an extract fromproliferating cells. While these data strongly suggest that p27^(Kip1)is at least a component of the Cdk2 inhibitor, it has not beendetermined whether inhibition is due to p27^(Kip1) alone, or whetherp27^(Kip1) recruits additional proteins to the cyclin E-Cdk2 complex. Ithas further been determined that p27, as well as cyclin E and Cdk2 arepresent in other organisms, e.g., mice and humans (data not shown).

The mechanism of p27^(Kip1) inhibition has features that distinguish itfrom pathways that control activation of the mitotic cyclin-Cdc2complexes. First, p27^(Kip1) appears to act stoichiometrically ratherthan catalytically. Second, tyrosine phosphorylation of Cdk2 was notdetected in inactive cyclin E-Cdk2 complexes containing p27^(Kip1),suggesting that p27^(Kip1) does not have tyrosine kinase activity orinhibit a tyrosine phosphatase. Complexes containing p27^(Kip1) were notefficiently phosphorylated by the p34Cdc2 activating kinase, CAK, andthis might be sufficient to explain their inactivity. It is possiblethat p27^(Kip1) dephosphorylates Thr160, although it would be surprisingif the enzymatic activity of a phosphatase were stable to heating to100° C. It is more likely that binding of p27^(Kip1) to the cyclinE-Cdk2 complex prevents Thr160 phosphorylation by altering theconformation of the T160 domain, or by sterically obstructing CAK. Itwould not be surprising if p27^(Kip1) functioned similarly to thenegative regulatory subunits or domains of other protein kinases,perhaps even interacting with the kinase active site as apseudosubstrate.

It is intriguing that in addition to p27^(Kip1) other potentialregulators of Cdk activity during G1 also bind directly to cyclin-Cdkcomplexes, including FAR1 (Peter et al., 1993), p40 (Mendenhall, 1993),p16 and p21 (Xiong et al., 1992; 1993) and Rb (Kato et al., 1993; Dowdyet al., 1993; Ewen et al., 1993a). While none of these other proteinshas yet been shown to directly inhibit Cdk activity, it seems likelythat at least some of them will perform this function. Directprotein-protein interactions may be a way to focus inhibitory signals onspecific cyclin-Cdk complexes in a cellular environment containing othermore promiscuous transacting regulators of Cdk activity.

p27^(Kip1) Links Growth Inhibitory Signals to Cell Cycle Arrest

p27^(Kip1) was discovered in cells arrested in G1 by either contactinhibition or TGF-β. A similar activity has also been found to blockCdk2 activation in various cell types deprived of specific growthfactors, including serum-starved fibroblasts and IL-2 deprivedlymphocytes (unpublished observations). Inhibition of Cdk2 activation byp27^(Kip1) or functionally similar proteins, may be a general mechanismthrough which diverse extracellular and intracellular signals exertcontrol on cell proliferation.

p27^(Kip1) constrains cell proliferation by setting the threshold levelof cyclin E necessary to activate Cdk2. If p27^(Kip1) actsstoichiometrically, as these data suggest, then the Cdk2 activationthreshold is reached soon after the amount of cyclin E in the cellexceeds the amount of active p27^(Kip1). In arrested cells thisthreshold is set higher than physiological cyclin E levels, andconsequently only inactive cyclin E-Cdk2 complexes assemble. The cyclinA-Cdk2 complex may be subject to similar control (Koff et al., 1993;Firpo et al., in preparation), and an inability to activate this complexshould also contribute to cell cycle arrest (Girard et al. 1991: PacJanoet al., 1992: 1993; Tsai et al., 1993).

How might growth inhibitory signals be linked to the activity ofp27^(Kip1)? The simplest idea would be that growing cells do not containmuch p27^(Kip1) and that signals which inhibit cell proliferation inducep27^(Kip1) synthesis or stabilization and thereby increase its amountabove a critical basal level. This model can not be strictly correctbecause greatly increased quantities of p27^(Kip1) can be recovered froma latent pool once extracts from prolifera-ting cells are subject toheat treatment. A substantial pool of p27^(Kip1) must be present inthese extracts and must be sequestered by other molecules. This impliesthat p27^(Kip1) plays a normal role during the proliferative cell cycleand is not simply a response element for signals which induce growtharrest. The abundance of “free” p27^(Kip1) that is able to interact withthe cyclin E-Cdk2 complex might, therefore, be modulated by an upstreamregulator, such as the cyclin D2-Cdk4 complex, which also binds top27^(Kip1) directly. This prevents association with cyclin E-Cdk2 andenables its functional activation, at least in vitro. The idea that p27activity is governed by an upstream regulator does not exclude thepossibility that the total cellular level of p27 may increase inarrested cells, and these experiments have not directly compared thetotal amounts of p27 in proliferating and arrested cells.

D-type cyclins are themselves targets of growth inhibitory signals(reviewed in Sherr, 1993). Their synthesis is rapidly reduced in growthfactor-deprived cells (Matsushime et al., 1991; Won et al., 1992; Katoet al., in press) and in contact inhibited cells (unpublishedobservations) leading to a reduction in cyclin D-Cdk4 levels (Matsushimeet al., 1992). While D-type cyclin levels are not greatly affected byTGF-β blockade, TGF-β does reduce synthesis of Cdk4 so that a netreduction in cyclin D-Cdk4 complexes is achieved nevertheless (Ewen etal., 1993b). In TGF-β inhibited cells, where Cdk4 is limiting,expression of excess Cdk4 should lead to the formation of additionalcyclin D-Cdk4 complexes and sequester p27^(Kip1). In fact, enforcedexpression of Cdk4 in vivo reverses the block to Cdk2 activation incells exposed to TGF-β (Ewen et al., 1993b). However, the addition ofCdk4 alone to extracts from TGF-β treated cells in vitro does notreverse the interaction of p27^(Kip1) with cyclin E-Cdk2. Unlikecomplexes with cyclin E and Cdk2, which can be formed in vitro by mixingthe recombinant proteins produced in insect cells, D-type cyclins andCdk4 do not assemble efficiently unless Sf9 cells are coinfected withbaculoviruses encoding both components (Kato et al., 1993). Although thereasons underlying these differences in complex formation have not beendefined, all results are internally consistent and support the idea thatcyclin D-Cdk4 complexes act upstream of cyclin E-Cdk2 by interactingwith p27^(Kip1). Although these ideas are based upon many observationsmade in intact cells, the proposed pathway containing cyclin D-Cdk4,p27^(Kip1) and cyclin E-Cdk2 has been tested directly only in vitro. Onemight expect that Cdk2 will be regulated by additional mechanisms, andthat other novel Cdk complexes in addition to cyclin D2-Cdk4 couldcontribute to the titration of p27^(Kip1).

It is not likely that the only role of cyclin D2-Cdk4 is to titratep27^(Kip1), but rather, complex accumulation is likely to trigger theCdk4-mediated phosphorylation of particular substrates necessary for G1progression. Thus cyclin D complexed with catalytically inactive Cdk4 issufficient to sequester p27^(Kip1), but is unlikely to fully substitutefor all essential Cdk4 functions in vivo.

One feature of p27^(Kip1) induced cell cycle arrest is that cells canaccumulate inactive cyclin E-Cdk2 complexes. Recovery from cell cyclearrest, therefore, might not require synthesis of new cyclin E andassembly of new cyclin E-Cdk2 complexes. Rather the cell may make use ofthis latent pool of inactive complexes when cell proliferation resumes.This might be essential under circumstances where the signals thatpromoted cyclin synthesis were transient, and absent when the growthinhibitory signals ceased. Thus far, however, conditions have not beendefined that allow re-activation of inactive cyclin E-Cdk2p27^(Kip1)complexes. In vitro, only cyclin E-Cdk2 complexes which assemble aftertitration of p27^(Kip1) are active, and the same may be true in vivo aswell.

The presence of p27^(Kip1) in proliferating cells suggests that its rolemay not be restricted to inducing cell cycle arrest in response toextracellular signals. It may also set the cyclin E threshold forexecution of the G1 to S transition during each mitotic cycle. Cellfusion experiments have indicated that entry into S phase in mammalianfibroblasts is controlled by an activator that accumulates continuouslyduring G1 (Foumier and Pardee, 1975; Rao et al., 1977). By comparing therate of S phase entry in mono- bi- and tri-nucleate cells it wasconcluded that the amount of this activator rather than itsconcentration was critical in determining the start of S phase. Theseobservations are consistent with a model in which the limiting step inCdk2 activation is not assembly of the cyclin-Cdk2 complex, which shouldbe concentration dependent, but instead involves the assembly of asufficient number of complexes to overcome a threshold level of astoichiometric inhibitor, such as p27^(Kip1). It is also pointed outthat spontaneous decay of p27^(Kip1) inhibited complexes to freep27^(Kip1) and active cyclin-Cdk2 might occur with first order(exponential) kinetics and could underlie the first order rate constantsfrequently reported for S phase entry in mammalian cells (Smith &Martin, 1973; Brooks et al., 1980).

p27^(Kip1) may Enforce Order During G1 Progression

Cyclin-Cdk complexes appear in a specific order as cells transit G1(Sherr, 1993). If it is assumed that this temporal order is essentialfor normal G1 progression, then cells must solve the problem ofrestoring order during recovery from cell cycle arrest. For example,contact inhibition and TGF-β interfere with the accumulation of cyclinD-Cdk4 complexes, but do not affect synthesis of cyclin E- and cyclinA-Cdk2 complexes, which act later in the cell cycle. If the cyclinE-Cdk2 and cyclin A-Cdk2 complexes were active during cell cycle arrest,then the normal order of Cdk action would be lost. p27^(Kip1) mightensure that this does not happen by preventing activation of thesepre-existing complexes during cell cycle arrest. In addition, if theactivity of p27^(Kip1) is itself controlled by cyclin D2-Cdk4, then thiswould provide an efficient mechanism for maintaining Cdk2 inactive untilcyclin D-Cdk4 complexes assemble and execute their functions.

II EXPERIMENTAL PROCEDURES

Metabolic Labeling, Immunoprecipitations and Peptide Mapping

Mv1Lu cells were synchronized by contact inhibition, treated with TGF-b,metabolically labeled, lysed, immunoprecipitated with anti-Cdk2 antibodyor chromatographed on cyclin E-Cdk2 affinity columns. For peptidemapping, the 27 kd band present in Cdk2 immunoprecipitates and in thecyclin E-Cdk2 affinity column eluates was cut out from the gels,digested with 0.1 μg of V8 protease, and resolved on 15-22.5% gradientgels.

Baculoviral Proteins

The human cyclin E cDNA (Koff et al., 1991) was tagged at the N-terminuswith a hexahistidine sequence. This cDNA was cloned into baculovirustransfer vector pVL1392, and expressed in Sf9 cells as described in theBaculoGold Transfection Kit (Pharmingen). Baculoviral proteins wereprepared by the method of Desai et al., 1992.

Kip1 Purification

Two hundred 150 mm dishes of contact inhibited Mv1Lu cells (˜2×10¹⁰cells) were collected by trypsinization and lysed in hypotonic buffer bysonication. The extracts were clarified by centrifugation, heated to100° C. for 5 min and clarified by centrifugation. Agarose-preclearedextracts were allowed to bind to His-cyclin E-Cdk2 complexes immobilizedon Ni⁺⁺-NTA-agarose. Specifically bound proteins were eluted with 6Mguanidium hydrochloride solution, dialyzed overnight against 1×HBBbuffer (25 mM HEPES-KOH, pH 7.7, 150 mM NaCl, 5 mM MgCl₂, 0.05% NP-40and 1 mM DTT) (Kaelin Jr et al., 1992) and acetone-precipitated.

Protein Sequence Analysis

Protein was fractionated by SDS-PAGE, electroblotted ontonitrocellulose, and the Ponceau S-stained 27 kDa band was excised andprocessed for internal amino acid sequence analysis (Tempst et al.,1990). HPLC peak fractions (over trypsin background) were analyzed by acombination of automated Edman degradation and matrix-assistedlaser-desorption (MALDI-TOF) mass spectrometry. Mass analysis (on 2%aliquots) was carried out using a model LaserTec Research MALDI-TOFinstrument (Vestec), and a-cyano-4-hydroxy cinnamic acid as the matrix.Chemical sequencing (on 95% of the sample) was done using an AppliedBiosystems 477A sequenator optimized for femtomole level analysis.

Kip1 cDNA Cloning and Northern Blot Analysis

RT-PCR reactions were performed using degenerate oligonucleotides asprimers and total RNA from contact-inhibited Mv1Lu cells as template.The combination of one pair of primers (see FIG. 9A) yielded a 135 bpfragment that was used to screen a lZAPII cDNA library prepared fromMv1Lu cells. The mouse Kip1 cDNA was obtained from a lEXlox mouse embryocDNA library (Novagen), and the human Kip1 cDNA was obtained from algt11 kidney cDNA library (Clontech). Poly(A)⁺ RNA blots were hybridizedwith a PCR-derived fragment of the mouse Kip1 cDNA labeled by randompriming.

In Vitro Translation

A NdeI-XhoI fragment containing the coding region of the mouse Kip1 cDNA(nucleotides 1-591) was subcloned to pCITE2a (Novagen). This constructencodes a fusion protein containing a C-terminal hexahistidine sequenceand 6 amino acids from the vector at the N-terminus of Kip1. In vitrotranscription and translation were performed using Red Nova lysate(Novagen).

Recombinant Kip1

A 591 bp PCR generated NheI-XhoI fragment of the mouse Kip1 cDNAcontaining the full length coding region was subloned into pET21a(Novagen), yielding a construct that encodes Kip1 with a C-terminalhexahistidine sequence. The protein was expressed in BL21 (DE3) bacteriaand purified by sonicating cells in a solution containing 8M urea, 50 mMTris-HCl (pH 7.4), 20 mM imidazole, clarified by centrifugation andbound to Ni⁺⁺-NTA agarose for 1 h at 4° C. The column was washed with a6M to 0.75 M urea reverse gradient in 0.5M sodium chloride, 50 mM Tris(pH 7.4), and 20% glycerol and eluted with 200 mM imidazole, 20 mM HEPESpH 7.4, 1M KCl, 100 mM EDTA. The eluate was dialysed overnight against1×HBB buffer and stored at −80° C. until use.

In Vitro Kinase and Cdk2 Activation Assays

H5 insect cell extracts containing baculovirally-expressed cyclins andCdks were incubated with recombinant Kip1 for 30 min at 37° C.,precipitated with anti-HA antibody, and the histone H1 kinase activityof these complexes was assayed (Koff et al., 1993). Rb kinase reactionswere done according to Matsushime et al. (1991). The phosphorylation ofthe histone H1 band and Rb band were quantitated with Phosphorimager(Molecular Dynamics).

Hypotonic cell extracts from exponentially growing A549 cells wereincubated with baculoviral His-cyclin E protein, with or without Kip1,at 37° C. for 30 min. Mixtures were then diluted 10-fold in 1×NP40 RIPAbuffer containing 20 mM imidazole and incubated with Ni⁺⁺-NTA-agarose at4° C. for 1 h. One portion of the samples was run on 12% SDS-PAGE, andimmunoblotted with anti-Cdk2 antibody (Koff et al., 1993).

Kip1 Transfections and Flow Cytometry Analysis

The mouse Kip1 cDNA (nucleotides −82 to +591) was subcloned into pCMV5(Attisano et al., 1993). R-1B cells were cotransfected with 0.5 μg/ml ofpCEXV-3 containing murine CD16 cDNA (Kurosaki and Ravetch, 1989) and 3μg/ml of pCMV5 alone, or with 3 μg/ml of pCMV5-Kip1 (Attisano et al.,1993). CD16 immunostained cells (Wirthmueller et al., 1992) wereanalyzed by flow cytometry using FACScan (Becton-Dickinson) andMulticycle software (PHOENIX Flow Systems).

Results

Purification and Cloning of Kip1

Lysates from contact-inhibited Mv1Lu cells were heated to 100° C.,cleared of insoluble material, and allowed to bind to a cyclin E-Cdk2affinity column. Elution with 6 M guanidium hydrochloride yieldedrecombinant Cdk2 released from the column, and the 27 kd protein Kip1.Dialyzed aliquots of this sample had strong inhibitory activity towardscyclin E-Cdk2 in histone H1 kinase assays, and this activity was shownto coelute from SDS-PAGE gel slices with Kip1. The Kip1 yield from twoseparate preparations (˜2×10¹⁰ cells each) was 0.3 μg and 1 μg,respectively.

In order to confirm that Kip1 interacts with Cdk2 in vivo, metabolicallylabeled extracts were immunoprecipitated from contact-inhibited Mv1Lucells using anti-Cdk2 antibodies (FIG. 8B). In addition to Cdk2, theprecipitate contained a 27 kd band whose peptide map, after limiteddigestion with V8 protease, was identical to that of Kip1 purified frommetabolically-labeled cells by cyclin E-Cdk2 affinity chromatography(FIG. 8C). These results provided further evidence that the Cdkinhibitor purified by binding to cyclin E-Cdk2 in vitro was associatedwith Cdk2 in quiescent cells.

Various Kip1 tryptic peptide sequences were obtained by automated Edmandegradation and used to design degenerate oligonucleotide primers forcDNA amplification by the reverse transcription-polymerase chainreaction (RT-PCR). A PCR product amplified out of reverse-transcribedMv1Lu mRNA was used to screen a Mv1Lu cDNA library. This yielded onesingle positive clone that encoded the sequences obtained from thepurified protein (FIG. 9A). Screening of cDNA libraries from humankidney and mouse embryo with the Kip1 cDNA yielded clones of highlyrelated sequence. The human and mouse Kip1 cDNAs (Genbank AccessionNumbers U10906 and U09968) had open reading frames of 594 and 591 bp,respectively, starting with an ATG codon in a favorable translationinitiation context and preceded by stop codons (data not shown).Compared to these open reading frames, the mink clone (Genbank AccessionNumber U09966) was incomplete, and ended at nucleotide 534 (FIG. 9A).

The Kip1 cDNA encodes a predicted protein of 198 amino acids (22,257daltons) in human and 197 amino acids (22,208 daltons) in mouse. Thesevalues are smaller than the 27 kd value obtained with the purified minkprotein by SDS-PAGE. To resolve this discrepancy, a cDNA encoding themouse Kip1 sequence was constructed, and tagged at the C-terminus with ahexahistidine sequence (˜1 kd mass). In vitro transcription andtranslation of this cDNA yielded a product that bound specifically toNi⁺⁺-NTA-agarose and migrated as a 28 kd protein on SDS-PAGE gels (FIG.8C), confirming that the cloned cDNA encodes full-length Kip1 and thatthis protein migrates on SDS-PAGE somewhat slower that its calculatedmolecular mass.

Kip1 is Highly Conserved and Related to Cip1/WAF1

The predicted human, mouse and mink Kip1 amino acid sequences are highlyrelated, showing ˜90% identity (FIG. 9A). A Genbank search revealedthat, at the amino acid level, Kip1 shows significant homology only toCip1/WAF1. The similarity was largely limited to a 60-amino acid segmentin the N-terminal half of the protein. This region was 44% identical tothe corresponding region in Cip1/WAF1 (FIG. 9B). Like Cip1/WAF1, Kip1has a putative bipartite nuclear localization signal (Dingwall andLaskey, 1991) near the C-terminus (FIG. 9B). Yet unlike Cip1/WAF1, theKip1 sequence does not have a putative zinc finger motif in theN-terminal region, and has a C-terminal extension of 23 amino acids thatcontains a consensus Cdc2 phosphorylation site (FIG. 9B).

Cdk Inhibitory Activity

Pure recombinant Kip1 tagged with hexahistidine at the C-terminusinhibited the histone H1 kinase activity of human cyclin A-Cdk2, cyclinE-Cdk2 and cyclin B1-Cdc2 complexes when assayed under linear reactionconditions (FIGS. 10A and 10B) whereas a mock sample from bacteriatransformed with vector alone did not. Cyclin E-Cdk2 was inhibitedhalf-maximally at 0.5 nM Kip1 (FIG. 10B). Complete inhibition of cyclinA-Cdk2 required an eight-fold higher concen-tration, and thisconcentration was not sufficient to completely block cyclin BD-Cdc2(FIG. 10B). Addition of Kip1 to cyclin E-Cdk2, cyclin A-Cdk2 or cyclinD2-Cdk4 complexes inhibited their ability to phosphorylate a GST-Rbfusion product (FIGS. 10C and D). The relative sensitivity of cyclinE-Cdk2 and cyclin A-Cdk2 to inhibition by Kip1 in these assaysparalleled their sensitivity in the histone H1 kinase assays (compareFIGS. 10B and 10D). Approximately 10 nM cyclin and 10 nM Cdk were usedin these assays, but the actual concentration of cyclin:Cdk complexes isnot known.

Cdk Inhibitory Domain

It was investigated whether the inhibitory activity of Kip1 resided inthe region of similarity to Cip1/WAF1. A 52-amino acid peptide [Kip1(28-79)] corresponding to this region in Kip1 (FIG. 10E) was producedrecombinantly and purified with a C-terminal hexahistidine tag. Thispeptide inhibited Rb phosphorylation by cyclin A-Cdk2 with a potencythat was close to that of full length Kip1 (FIG. 10E) and inhibitedcyclin E-Cdk2 or cyclin D2:Cdk4 less effectively. Versions of this Kip1region missing three amino acids at the N-terminus or fifteen at theC-terminus, were much weaker as Cdk inhibitors, and deletion of sevenN-terminal amino acids yielded a product with no inhibitory activity(FIG. 10E). The peptide Kip1 [(104-152)] which has little sequencesimilarity to Cip1/WAF1, was inactive as a Cdk inhibitor (FIG. 10E).

Kip1 Prevents Cdk2 Activation

Kip1 was originally identified as a factor whose presence in extractsfrom quiescent cells rendered Cdk2 refractory to activation byphosphorylation at Thr¹⁶⁰. In order to determine if Kip1 could block Cdkactivation, its effect on cyclin E-dependent Cdk2 activation in extractsfrom exponentially growing cells was assayed (Koff et al., 1993). A549human lung carcinoma cell extracts were incubated with histidine-taggedcyclin E which was then retrieved and assayed for associated histone H1kinase activity (FIG. 11A). Addition of histidine-tagged Kip1 to thecell extracts markedly decreased the level of cyclin E-associated kinaseactivity (FIG. 11A). In parallel assays, the retrieved cyclin E wassubjected to SDS-PAGE and western blotting with anti-Cdk2 antibodies.Cell extracts that did not receive Kip1 yielded cyclin E-associated Cdk2in a form that corresponds to Cdk2 phosphorylated at Thr¹⁶⁰ (Gu et al.,1992) (FIG. 11B). In contrast, cyclin E-associated Cdk2 from extractsthat received Kip1 was exclusively in the inactive form (FIG. 11B).Collectively, these results suggested that Kip1 binding to preactivecyclin E-Cdk2 complexes in vitro prevented Thr¹⁶⁰ phosphorylation andactivation of Cdk2.

Kip1 Overexpression Inhibits Cell Entry Into S Phase

Mouse Kip1 subcloned into a mammalian expression vector was transfectedinto Mv1Lu cells under conditions in which up to 656 of the cellpopulation takes up and transiently expresses transfected plasmids(Attisano et al., 1993). The rate of ¹²⁵I-deoxyuridine incorporationinto DNA was reduced 70% in cells transfected with Kip1 compared tocells transfected with vector alone (Table III).

TABLE III Kip1 blocks entry into S phase Time after CD16⁺¹²⁵I-deoxyuridine Percentage of transfection Vector incorporation^(a)cells in S phase^(b) 24 h pCMV5 27,581 ± 5,126  26 ± 5  pCMV5-Kip1 8,386± 1,250 9 ± 2 43 h pCMV5 5,126 ± 47   35 ± 2  pCMV5-Kip1 1,510 ± 140   7± 1 The TGF-b receptor-defective R-1B cell line was cotransfected with ahuman CD16 expression vector and pCMV5 or pCMV5 containing the mouseKip1 cDNA. Assays were conducted at the indicated times aftertransfections. ^(a125)I-deoxy-uridine incorporated over a 3 h period bythe entire cell population. Data are the average ± S.D. of triplicatedeterminations. ^(b)Transfected cells were immunostained with anti-CD16and analyzed for DNA content. Data are the average of two separateexperiments, and show the range of values.

To determine the effect on cell cycle distribution, Kip1 wascotransfected with a CD16 expression vector (Kurosaki and Ravetch, 1989)that allowed flow cytometric separation of the transfected cells basedon CD16 immunofluorescence. The CD16⁺ population cotransfected with Kip1showed a larger proportion of cells in G1 phase and a smaller proportionin S phase than the CD16⁺ population cotransfected with vector alone(Table III), suggesting that Kip1 overexpression obstructed cell entryinto S phase. Cell numbers after transfection indicated that Kip1 didnot cause cell death (data not shown).

Kip1 mRNA Distribution and Levels in Quiescent and Proliferating Cells

The level of endogenous Kip1 mRNA expression in various human tissueswas determined by Northern blot analysis. The only mRNA detected was aspecies of 2.5 kb present at similar levels in all tissues tested,although it was somewhat higher in skeletal muscle and lower in liverand kidney (FIG. 12A). Kip1 mRNA levels were similar in exponentiallyproliferating and contact-inhibited Mv1Lu cells, and did not change whencells were released from contact inhibition by being plated at lowdensity in the presence of serum (FIG. 12B). Addition of TGF-b to cellsreleased from contact-inhibition also did not affect Kip1 mRNA levels(FIG. 12B). These results indicate that the regulation of Kip1 byextracellular antiproliferative signals occurs at a post-transcriptionallevel.

Discussion

A Family of Cdk Inhibitors

Human Kip1 encodes a protein of 198 amino acids that is highly conserved(˜90% identity) in mouse and mink. Its most distinctive feature is a60-amino acid region in the N-terminal half that has amino acid sequencesimilarity to Cip1/WAF1 (El-Deiry et al., 1993; Harper et al., 1993;Xiong et al., 1993). Like Cip1/WAF1, Kip1 contains a potential nuclearlocalization signal in the C-terminal region. In Kip1, this region alsocontains a consensus Cdc2 kinase site that might play a role infeed-back regulation by their target kinases.

The structural similarity between Kip1 and Cip1/WAF1 defines a family ofmammalian Cdk inhibitors with different regulatory properties. Kip1 isinvolved post-transcription-ally in the action of extracellular signals(present work) and its silencing in exponentially growing cellscorrelates with binding to a heat-labile component. In contrast,Cip1/WAF1 is regulated transcriptionally by p53, senescence and cellquiescence. Kip1 and Cip1/WAF1 are more effective against G1 Cdks thanagainst mitotic Cdks. However, Kip1 was more effective against cyclinE-Cdk2 than against cyclin A-Cdk2 (or cyclin D2-Cdk4) whereas in similarassays, Cip1/WAF1 was more effective against cyclin A-Cdk2 (Harper etal., 1993). Kip1 effectiveness is likely defined by its binding affinityfor a given cyclin-Cdk complex.

The Kip1 region that is similar to Cip1/WAF1 is sufficient to inhibitCdk activity when tested as a 52-amino acid peptide in vitro. This 52amino acid segment contains the sequence LFGPVN (SEQ ID NO: 22) whichcorresponds to the longest un-interrupted stretch of identity toCip1/WAF1 and, interestingly, is similar to the FAR1 sequence LSQPVN(SEQ ID NO: 23) located in a region required for interaction withCLN2-CDC28 (Peter et al., 1993).

Cdk Inhibition at Two Levels

Kip1 can inhibit both the process of Cdk activation and the kinaseactivity of cyclin-Cdk complexes assembled and activated in intactcells. Kip1 was originally identified as a factor whose presence inextracts of quiescent cells rendered them unable to activate Cdk2 byphosphorylation at Thr¹⁶⁰. Indeed, recombinant Kip1 inhibits Cdk2 Thr¹⁶⁰phosphorylation and activation in vitro. Although Kip1 could act as aninhibitor of the Cdk-activating kinase, previous results tend to argueagainst this possibility. The dual effects of Kip1, both on Cdk2activation and Cdk2 activity, might relate to the fact that Thr¹⁶⁰ islocated in a loop that closes the substrate-binding cleft in the Cdk2structure (DeBondt et al., 1993). It is conceivable that binding of Kip1to this region might interfere with Thr¹⁶⁰ phosphorylation as well aswith the catalytic function of activated Cdk2.

Function in the Cell Cycle

Cyclin E-Cdk2 and cyclin D-Cdk4 are rate limiting for G1 progression(Jiang et al., 1993; Ohtsubo and Roberts, 1993; Quelle et al., 1993).Inhibition of these kinases by Kip1 in vivo would render cells unable toreach that transition. The strong reductions in the rate of DNAsynthesis and the proportion of cells in S phase caused by Kip1transfection are consistent with this possibility and with a role ofKip1 as mediator of extracellular growth inhibitory signals.

As cells released from contact inhibition move closer to S phase, theirextracts contain progressively lower levels of Kip1 activity, and thisdecline can be prevented by TGF-b addition early in G1 phase. However,the present results show that contact-inhibited cells and TGF-b-treatedcells have Kip1 mRNA levels equal to those of proliferating cells.Furthermore, extracts from proliferating cells yield active Kip1 whenthey are heated transiently at 100° C. One interpretation of theseobservations is that Kip1 is progressively sequestered by binding to aheat-labile component as cells progress through G1, and this process canbe prevented by TGF-b. Mitogens and antimitogens might regulate Kip1activity or availability by controlling its binding to a silencingprotein. Alternatively, Kip1 might be a passive regulator whose uniformlevels could ensure that active Cdks become available only when theirlevels reach the threshold imposed by binding to Kip1. In the lattersituation, even small effects of mitogens and antimitogens on cyclin orCdk protein levels could become amplified by the existence of thatthreshold.

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27 596 base pairs nucleic acid single linear cDNA CDS 1..594 1 ATG TCAAAC GTG CGA GTG TCT AAC GGG AGC CCT AGC CTG GAG CGG ATG 48 Met Ser AsnVal Arg Val Ser Asn Gly Ser Pro Ser Leu Glu Arg Met 1 5 10 15 GAC GCCAGG CAG GCG GAG CAG CCC AAG CCC TCG GCC TGC AGG AAC CTC 96 Asp Ala ArgGln Ala Glu Gln Pro Lys Pro Ser Ala Cys Arg Asn Leu 20 25 30 TTC GGC CCGGTG GAC CAC GAA GAG TTA ACC CGG GAC TTG GAG AAG CAC 144 Phe Gly Pro ValAsp His Glu Glu Leu Thr Arg Asp Leu Glu Lys His 35 40 45 TGC AGA GAC ATGGAA GAG GCG AGC CAG CGC AAG TGG AAT TTC GAT TTT 192 Cys Arg Asp Met GluGlu Ala Ser Gln Arg Lys Trp Asn Phe Asp Phe 50 55 60 CAG AAT CAC AAA CCCCTA GAG GGC AAG TAC GAG TGG CAA GAG GTG GAG 240 Gln Asn His Lys Pro LeuGlu Gly Lys Tyr Glu Trp Gln Glu Val Glu 65 70 75 80 AAG GGC AGC TTG CCCGAG TTC TAC TAC AGA CCC CCG CGG CCC CCC AAA 288 Lys Gly Ser Leu Pro GluPhe Tyr Tyr Arg Pro Pro Arg Pro Pro Lys 85 90 95 GGT GGC TGC AAG GTG CCGGCG CAG GAG AGC CAG GAT GTC AGC GGG AGC 336 Gly Gly Cys Lys Val Pro AlaGln Glu Ser Gln Asp Val Ser Gly Ser 100 105 110 CGC CCG GCG GCG CCT TTAATT GGG GCT CCG GCT AAC TCT GAG GAC ACG 384 Arg Pro Ala Ala Pro Leu IleGly Ala Pro Ala Asn Ser Glu Asp Thr 115 120 125 CAT TTG GTG GAC CCA AAGACT GAT CCG TCG GAC AGC CAG ACG GGG TTA 432 His Leu Val Asp Pro Lys ThrAsp Pro Ser Asp Ser Gln Thr Gly Leu 130 135 140 GCG GAG CAA TGC GCA GGAATA AGG AAG CGA CCT GCA ACC GAC GAT TCT 480 Ala Glu Gln Cys Ala Gly IleArg Lys Arg Pro Ala Thr Asp Asp Ser 145 150 155 160 TCT ACT CAA AAC AAAAGA GCC AAC AGA ACA GAA GAA AAT GTT TCA GAC 528 Ser Thr Gln Asn Lys ArgAla Asn Arg Thr Glu Glu Asn Val Ser Asp 165 170 175 GGT TCC CCA AAT GCCGGT TCT GTG GAG CAG ACG CCC AAG AAG CCT GGC 576 Gly Ser Pro Asn Ala GlySer Val Glu Gln Thr Pro Lys Lys Pro Gly 180 185 190 CTC AGA AGA CGT CAAACG TA 596 Leu Arg Arg Arg Gln Thr 195 198 amino acids amino acid linearprotein 2 Met Ser Asn Val Arg Val Ser Asn Gly Ser Pro Ser Leu Glu ArgMet 1 5 10 15 Asp Ala Arg Gln Ala Glu Gln Pro Lys Pro Ser Ala Cys ArgAsn Leu 20 25 30 Phe Gly Pro Val Asp His Glu Glu Leu Thr Arg Asp Leu GluLys His 35 40 45 Cys Arg Asp Met Glu Glu Ala Ser Gln Arg Lys Trp Asn PheAsp Phe 50 55 60 Gln Asn His Lys Pro Leu Glu Gly Lys Tyr Glu Trp Gln GluVal Glu 65 70 75 80 Lys Gly Ser Leu Pro Glu Phe Tyr Tyr Arg Pro Pro ArgPro Pro Lys 85 90 95 Gly Gly Cys Lys Val Pro Ala Gln Glu Ser Gln Asp ValSer Gly Ser 100 105 110 Arg Pro Ala Ala Pro Leu Ile Gly Ala Pro Ala AsnSer Glu Asp Thr 115 120 125 His Leu Val Asp Pro Lys Thr Asp Pro Ser AspSer Gln Thr Gly Leu 130 135 140 Ala Glu Gln Cys Ala Gly Ile Arg Lys ArgPro Ala Thr Asp Asp Ser 145 150 155 160 Ser Thr Gln Asn Lys Arg Ala AsnArg Thr Glu Glu Asn Val Ser Asp 165 170 175 Gly Ser Pro Asn Ala Gly SerVal Glu Gln Thr Pro Lys Lys Pro Gly 180 185 190 Leu Arg Arg Arg Gln Thr195 593 base pairs nucleic acid single linear cDNA CDS 1..591 3 ATG TCAAAC GTG AGA GTG TCT AAC GGG AGC CCG AGC CTG GAG CGG ATG 48 Met Ser AsnVal Arg Val Ser Asn Gly Ser Pro Ser Leu Glu Arg Met 1 5 10 15 GAC GCCAGA CAA GCG GAT CAC CCC AAG CCT TCC GCC TGC AGA AAT CTC 96 Asp Ala ArgGln Ala Asp His Pro Lys Pro Ser Ala Cys Arg Asn Leu 20 25 30 TTC GGC CCGGTC AAT CAT GAA GAA CTA ACC CGG GAC TTG GAG AAG CAC 144 Phe Gly Pro ValAsn His Glu Glu Leu Thr Arg Asp Leu Glu Lys His 35 40 45 TGC CGG GAT ATGGAA GAA GCG AGT CAG CGC AAG TGG AAT TTC GAC TTT 192 Cys Arg Asp Met GluGlu Ala Ser Gln Arg Lys Trp Asn Phe Asp Phe 50 55 60 CAG AAT CAT AAG CCCCTG GAG GGC AGA TAC GAA TGG CAG GAG GTG GAG 240 Gln Asn His Lys Pro LeuGlu Gly Arg Tyr Glu Trp Gln Glu Val Glu 65 70 75 80 AGG GGC AGC TTG CCCGAG TTC TAC TAC AGG CCC CCG CGC CCC CCC AAG 288 Arg Gly Ser Leu Pro GluPhe Tyr Tyr Arg Pro Pro Arg Pro Pro Lys 85 90 95 AGC GCC TGC AAG GTG CTGGCG CAG GAG AGC CAG GAT GTC AGC GGG AGC 336 Ser Ala Cys Lys Val Leu AlaGln Glu Ser Gln Asp Val Ser Gly Ser 100 105 110 CGC CAG GCG GTG CCT TTAATT GGG TCT CAG GCA AAC TCT GAG GAC CGG 384 Arg Gln Ala Val Pro Leu IleGly Ser Gln Ala Asn Ser Glu Asp Arg 115 120 125 CAT TTG GTG GAC CAA ATGCCT GAC TCG TCA GAC ATT CAG GCT GGG TTA 432 His Leu Val Asp Gln Met ProAsp Ser Ser Asp Ile Gln Ala Gly Leu 130 135 140 GCG GAG CAG TGT CCA GGGATG AGG AAG CGA CCT GCT GCA GAA GAT TCT 480 Ala Glu Gln Cys Pro Gly MetArg Lys Arg Pro Ala Ala Glu Asp Ser 145 150 155 160 TCT TCG CAA AAC AAAAGG GCC AAC AGA ACA GAA GAA AAT GTT TCA GAC 528 Ser Ser Gln Asn Lys ArgAla Asn Arg Thr Glu Glu Asn Val Ser Asp 165 170 175 GGT TCC CCG AAC GCTGGC ACT GTG GAG CAG ACG CCC AAG AAG CCC GGC 576 Gly Ser Pro Asn Ala GlyThr Val Glu Gln Thr Pro Lys Lys Pro Gly 180 185 190 CTT CGA CGC CAG ACGTA 593 Leu Arg Arg Gln Thr 195 197 amino acids amino acid linear protein4 Met Ser Asn Val Arg Val Ser Asn Gly Ser Pro Ser Leu Glu Arg Met 1 5 1015 Asp Ala Arg Gln Ala Asp His Pro Lys Pro Ser Ala Cys Arg Asn Leu 20 2530 Phe Gly Pro Val Asn His Glu Glu Leu Thr Arg Asp Leu Glu Lys His 35 4045 Cys Arg Asp Met Glu Glu Ala Ser Gln Arg Lys Trp Asn Phe Asp Phe 50 5560 Gln Asn His Lys Pro Leu Glu Gly Arg Tyr Glu Trp Gln Glu Val Glu 65 7075 80 Arg Gly Ser Leu Pro Glu Phe Tyr Tyr Arg Pro Pro Arg Pro Pro Lys 8590 95 Ser Ala Cys Lys Val Leu Ala Gln Glu Ser Gln Asp Val Ser Gly Ser100 105 110 Arg Gln Ala Val Pro Leu Ile Gly Ser Gln Ala Asn Ser Glu AspArg 115 120 125 His Leu Val Asp Gln Met Pro Asp Ser Ser Asp Ile Gln AlaGly Leu 130 135 140 Ala Glu Gln Cys Pro Gly Met Arg Lys Arg Pro Ala AlaGlu Asp Ser 145 150 155 160 Ser Ser Gln Asn Lys Arg Ala Asn Arg Thr GluGlu Asn Val Ser Asp 165 170 175 Gly Ser Pro Asn Ala Gly Thr Val Glu GlnThr Pro Lys Lys Pro Gly 180 185 190 Leu Arg Arg Gln Thr 195 534 basepairs nucleic acid single linear cDNA CDS 1..534 5 ATG TCA AAC GTG CGGGTG TCT AAC GGG AGC CCG AGC CTG GAG CGG ATG 48 Met Ser Asn Val Arg ValSer Asn Gly Ser Pro Ser Leu Glu Arg Met 1 5 10 15 GAC GCC AGA CAG GCGGAG TAC CCC AAG CCC TCC GCC TGC AGA AAC CTC 96 Asp Ala Arg Gln Ala GluTyr Pro Lys Pro Ser Ala Cys Arg Asn Leu 20 25 30 TTC GGC CCG GTC AAC CACGAA GAG CTG ACC CGG GAC TTG GAG AAG CAC 144 Phe Gly Pro Val Asn His GluGlu Leu Thr Arg Asp Leu Glu Lys His 35 40 45 CGC AGA GAC ATG GAA GAG GCAAGC CAG CGC AAG TGG AAT TTT GAT TTC 192 Arg Arg Asp Met Glu Glu Ala SerGln Arg Lys Trp Asn Phe Asp Phe 50 55 60 CAG AAT CAC AAG CCC CTG GAG GGCAAA TAC GAG TGG CAG GAG GTG GAG 240 Gln Asn His Lys Pro Leu Glu Gly LysTyr Glu Trp Gln Glu Val Glu 65 70 75 80 AAG GGC AGC TTG CCG GAG TTC TACTAC AGA CCC CCG CGG CCA CCC AAA 288 Lys Gly Ser Leu Pro Glu Phe Tyr TyrArg Pro Pro Arg Pro Pro Lys 85 90 95 GGC GCC TGC AAG GTG CCG GCG CAG GAGAGC CAG GAC GTC AGC GGG ACC 336 Gly Ala Cys Lys Val Pro Ala Gln Glu SerGln Asp Val Ser Gly Thr 100 105 110 CGG CAG GCC GTG CCT TTA ATG GGG TCTCAG GCA AAC TCA GAG GAC ACA 384 Arg Gln Ala Val Pro Leu Met Gly Ser GlnAla Asn Ser Glu Asp Thr 115 120 125 CAC TTG GTA GAC CAA AAG ACT GAC ACGGCG GAC AAC CAG GCT GGC TTA 432 His Leu Val Asp Gln Lys Thr Asp Thr AlaAsp Asn Gln Ala Gly Leu 130 135 140 GCG GAG CAG TGC ACT GGG ATC AGG AAGCGA CCG GCC ACA GAC GAT TCC 480 Ala Glu Gln Cys Thr Gly Ile Arg Lys ArgPro Ala Thr Asp Asp Ser 145 150 155 160 TCT CCT CAA AAC AAA AGA GCC AACAGA ACA GAA GAA AAT GTC TCA GAC 528 Ser Pro Gln Asn Lys Arg Ala Asn ArgThr Glu Glu Asn Val Ser Asp 165 170 175 GGT TCC 534 Gly Ser 178 aminoacids amino acid linear protein 6 Met Ser Asn Val Arg Val Ser Asn GlySer Pro Ser Leu Glu Arg Met 1 5 10 15 Asp Ala Arg Gln Ala Glu Tyr ProLys Pro Ser Ala Cys Arg Asn Leu 20 25 30 Phe Gly Pro Val Asn His Glu GluLeu Thr Arg Asp Leu Glu Lys His 35 40 45 Arg Arg Asp Met Glu Glu Ala SerGln Arg Lys Trp Asn Phe Asp Phe 50 55 60 Gln Asn His Lys Pro Leu Glu GlyLys Tyr Glu Trp Gln Glu Val Glu 65 70 75 80 Lys Gly Ser Leu Pro Glu PheTyr Tyr Arg Pro Pro Arg Pro Pro Lys 85 90 95 Gly Ala Cys Lys Val Pro AlaGln Glu Ser Gln Asp Val Ser Gly Thr 100 105 110 Arg Gln Ala Val Pro LeuMet Gly Ser Gln Ala Asn Ser Glu Asp Thr 115 120 125 His Leu Val Asp GlnLys Thr Asp Thr Ala Asp Asn Gln Ala Gly Leu 130 135 140 Ala Glu Gln CysThr Gly Ile Arg Lys Arg Pro Ala Thr Asp Asp Ser 145 150 155 160 Ser ProGln Asn Lys Arg Ala Asn Arg Thr Glu Glu Asn Val Ser Asp 165 170 175 GlySer 10 amino acids amino acid <Unknown> linear peptide 7 Asn Leu Tyr ProLeu Thr Asn Tyr Thr Phe 1 5 10 13 amino acids amino acid <Unknown>linear peptide 8 Thr Asp Thr Ala Asp Asn Gln Ala Gly Leu Ala Glu Gln 1 510 10 amino acids amino acid <Unknown> linear peptide 9 Gln Ala Val ProLeu Met Gly Pro Gln Glu 1 5 10 12 amino acids amino acid <Unknown>linear peptide 10 Leu Pro Glu Phe Tyr Tyr Arg Pro Pro Arg Pro Pro 1 5 106 amino acids amino acid <Unknown> linear peptide 11 Tyr Glu Trp Gln GluVal 1 5 20 base pairs nucleic acid single linear other nucleic acid/desc = “oligonucleotide” 12 ACNGAYACNG AYAAYCARGC 20 24 base pairsnucleic acid single linear other nucleic acid /desc = “oligonucleotide”YES 13 NGCYTGRTTR TCNGCNGTRT CNGT 24 20 base pairs nucleic acid singlelinear other nucleic acid /desc = “oligonucleotide” 14 CARGCNGTNCCNCTNATGGG 20 20 base pairs nucleic acid single linear other nucleicacid /desc = “oligonucleotide” 15 CARGCNGTNC CNTTRATGGG 20 21 base pairsnucleic acid single linear other nucleic acid /desc = “oligonucleotide”YES 16 NCCCATNAGN GGNACNGCYT G 21 21 base pairs nucleic acid singlelinear other nucleic acid /desc = “oligonucleotide” YES 17 NCCCATYAANGGNACNGCYT G 21 17 base pairs nucleic acid single linear other nucleicacid /desc = “oligonucleotide” 18 CCNGARTTYT AYTAYMG 17 17 base pairsnucleic acid single linear other nucleic acid /desc = “oligonucleotide”YES 19 CKRTARTARA AYTCNGG 17 17 base pairs nucleic acid single linearother nucleic acid /desc = “oligonucleotide” 20 TAYGARTGGC ARGARGT 17 18base pairs nucleic acid single linear other nucleic acid /desc =“oligonucleotide” 21 NACYTCYTGC CAYTCRTA 18 6 amino acids amino acid<Unknown> linear peptide 22 Leu Phe Gly Pro Val Asn 1 5 6 amino acidsamino acid <Unknown> linear peptide 23 Leu Ser Gln Pro Val Asn 1 5 164amino acids amino acid <Unknown> linear protein 24 Met Ser Glu Pro AlaGly Asp Val Arg Gln Asn Pro Cys Gly Ser Lys 1 5 10 15 Ala Cys Arg ArgLeu Phe Gly Pro Val Asp Ser Glu Gln Leu Ser Arg 20 25 30 Asp Cys Asp AlaLeu Met Ala Gly Cys Ile Gln Glu Ala Arg Glu Arg 35 40 45 Trp Asn Phe AspPhe Val Thr Glu Thr Pro Leu Glu Gly Asp Phe Ala 50 55 60 Trp Glu Arg ValArg Gly Leu Gly Leu Pro Lys Leu Tyr Leu Pro Thr 65 70 75 80 Gly Pro ArgArg Gly Arg Asp Glu Leu Gly Gly Gly Arg Arg Pro Gly 85 90 95 Thr Ser ProAla Leu Leu Gln Gly Thr Ala Glu Glu Asp His Val Asp 100 105 110 Leu SerLeu Ser Cys Thr Leu Val Pro Arg Ser Gly Glu Gln Ala Glu 115 120 125 GlySer Pro Gly Gly Pro Gly Asp Ser Gln Gly Arg Lys Arg Arg Glu 130 135 140Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 145 150155 160 Lys Arg Lys Pro 6 amino acids amino acid <Unknown> linearpeptide 25 Leu Phe Gly Pro Val Asp 1 5 13 amino acids amino acid<Unknown> linear peptide 26 Asn Leu Phe Gly Pro Val Asn His Glu Glu LeuThr Arg 1 5 10 13 amino acids amino acid <Unknown> linear peptide 27 AsnLeu Phe Gly Pro Val Asp His Glu Glu Leu Thr Arg 1 5 10

What is claimed is:
 1. An isolated nucleic acid comprising a nucleotidesequence represented in SEQ ID No.
 1. 2. An isolated nucleic acidcomprising a coding sequence for a polypeptide, which polypeptideincludes an amino acid sequence represented in SEQ ID No.
 2. 3. Anisolated nucleic acid comprising a nucleotide sequence encoding apolypeptide including an amino acid portion at least 90% identical toresidues +28 to +88 of SEQ ID No. 2, wherein said amino acid portioninhibits activation of a mammalian cyclin E-Cdk2 complex.
 4. A nucleicacid comprising: (i) a nucleotide sequence encoding a polypeptide thatinhibits activation of a mammalian cyclin E-Cdk2 complex, whichpolypepuide includes a portion at least 90% identical to residues +28 to+88 of SEQ ID No. 2; and (ii) a heterologous transcriptional regulatorysequence operably linked to the coding sequence.
 5. The nucleic acid ofclaim 3, wherein said polypeptide comprises an amino acid sequenceselected from SEQ ID No. 2, 4 or
 6. 6. The nucleic acid of claim 4,wherein said polypeptide comprises an amino acid sequence selected fromSEQ ID No. 2, 4 or
 6. 7. A nucleic acid encoding a polypeptide having anamino acid sequence consisting essentially of residues 28-79 of SEQ IDNo. 2, 4 or
 6. 8. The nucleic acid of claim 3 or 4, wherein thepolypeptide encoded by the nucleic acid includes an amino acid sequencecorresponding to residues 28-79 of SEQ ID No.
 2. 9. The nucleic acid ofclaim 3 or 4, wherein the polypeptide is a fusion protein.
 10. Thenucleic acid of claim 3 or 4, wherein the polypeptide inhibits cellcycle progression.
 11. The nucleic acid of claim 3, wherein thepolypeptide has a molecular weight of about 27 kD.
 12. The nucleic acidof claim 3, further comprising a transcriptional regulatory sequenceoperably linked to said nucleotide sequence.
 13. An expression vector,capable of replicating in a prokaryotic cell or a eukaryotic cell,comprising the nucleic acid of claim
 3. 14. The expression vector ofclaim 13, which vector is a viral vector.
 15. A host cell transfectedwith the expression vector of claim 13, and expressing said polypeptide.16. A method of producing a recombinant cell-cycle regulatory proteincomprising culturing the cell of claim 15 in a cell culture medium toexpress said polypeptide and isolating said polypeptide from said cellculture, thereby producing the recombinant cell-cycle regulatoryprotein.
 17. A nucleic acid composition comprising the nucleic acid ofclaim
 3. 18. The nucleic acid of claim 3, wherein said polypeptide is amammalian polypeptide.
 19. The nucleic acid of claim 1, wherein saidpolypeptide is a human polypeptide.